Julia Burel

Emily Williamson

Increasing cheetah population with CRISPR/Cas9 genetic engineering via electroporation method vs. pronuclear
injection method

I. Introduction
Background
Around 12,000 years ago, the population of African cheetah (Acinonyx jubatus) experienced a
bottleneck which resulted in a monophyletic species with genetic uniformity among a variety of loci (Menotti-
Raymond and O’Brien 1992). The cause of this bottleneck is unclear but it is believed to have occurred in from
the late Pleistocene (circa 10,000 years ago) when other species of cheetahs went extinct (O’Brien et al. 1987).
Before the bottleneck, there existed at least four paleontological and four subspecies of cheetahs that ranged
among four continents: North America, Africa, Asia and Europe (Menotti-Raymond and O’Brien 1992).
Currently, there are around 6,000 cheetahs left in the wild and the population is actively decreasing
(IUCN 2015). Ranges are primarily found in southern, eastern, and north-western Africa, with a smaller
population of Asiatic cheetah (Acinonyx jubatus venaticus) located in Iran (IUCN 2015, Mohammad et al. 2017).
Even though cheetahs tend to hunt smaller prey, they are often in interspecific competition for prey
with other large carnivores such as leopards (Panthera pardus) and lions (Panthera leo). Although females are
generally solitary, males will form groups called coalitions and can hunt larger prey. Larger carnivores have also
taken advantage of the smaller, normally solitary cheetah by stealing kills and killing cubs (Broekhuis et al.
2018). Other general threats to the species include hunting, trapping, livestock farming, and diseases (IUCN
2015).
The main threat to this species is the population’s low genetic diversity due to a deleterious gene that
creates a low semen quality. This arose through inbreeding after suffering a second bottleneck that began
within the last century and resulted in the loss of alleles (Terrell et al. 2016; O’Brien et al. 1987). While one
study suggests that low semen quality does not correlate with low heterozygosity, it is possible that because
male cheetahs produce lower amounts of spermatozoa this might prevent successful fertilization (Terrell et al.
2016). The intent of this study is to aid in increasing the population of the endangered and genetically deficient
African cheetah. Research is needed for increasing cheetah population trends, as well as identifying genetic
deficiencies within the species (IUCN 2015). This study will contribute to these needs by determining fitness
after genetic modification of embryos either by electroporation or pronuclear injection. A selective breeding
program will also be enacted which will be used to monitor the success rate of the two techniques. Any data
 gathered after the release of the modified individuals would help determine how the modified species interacts
with its environment and what areas must be managed to help conserve the population.

Objectives
The objectives of this study are to increase the declining population of cheetahs by introducing more
genetic diversity, and to determine if there is a correlation between genetic diversity and a stable population.
The population will be considered stable when numbers are not only actively increasing, but maintained. This
study also aims to determine which CRSIPR/Cas9 method is better at producing viable, successful offspring:
electroporation or pronuclear injection. We hypothesize that the genetically engineered cheetahs will have a
longer lifespan than wild cheetahs, as well as produce more genetically diverse, successful offspring.

II. Literature Review

The main threat to cheetah survival is the population’s low genetic diversity and subsequent inbreeding. While
the management of captive cheetah is primarily concerned with reducing inbreeding, this does not factor in the eroding
genetic diversity (Terrell et al. 2016). Breeding programs, such as the successful San Diego Safari Park which has
experienced 157 cheetah births, while still important in maintaining cheetah populations, cannot produce a stable wild
population (San Diego Zoo 2019). Even if these programs could produce thousands of cheetahs, we can assume that
nothing would change as their lack in genetic diversity would remain, and once released back into the wild, it is likely
the population would experience another severe crash. Therefore, this study is necessary to determine not only the
survival rate of genetically modified cheetahs, but if those cheetahs can provide population stability as well via
increased fitness.
A way to introduce genetic diversity to an organism is through the gene-editing technology, CRISPR/Cas9.
Prokaryotes use CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) as a memory based immune
system that can be passed down to the next generation. Because CRISPR is a heritable immune system, memories of
previous viral infections are provided to prokaryotes and a defense is built to detect and destroy returning viruses by
targeting and cleaving them using the Cas protein, an endonuclease (Memi et al. 2018). The system also has a short
noncoding guide RNA (gRNA) that helps to locate the target sequence and allows Cas9 to bind and initiate a double
strand break. After the break is completed, the DNA is repaired either through non-homologous end joining (NHEJ) or
homology-directed repair (HDR). Though HDR is more precise, NHEJ is more commonly utilized as it is more efficient
(Memi et al. 2018). Since its discovery, CRISPR/Cas9 has been developed into a low-cost, efficient gene-editing tool with
proven research applications.
One such application was enacted on mice with the intent to analyze essential genes for male fertility by
genetically altering coat colors (Abbasi et al. 2018). Four CRISPR/Cas9 methods were implemented: the embryonic stem
cell method, cytoplasmic injection, pronuclear injection, and electroporation. The embryonic stem cell method was
developed by mutating embryonic stem cells and injecting them into the blastocysts of wild-type mice to produce
chimeric mice. However, when the chimeric mice were bred together to produce the desired mutation, many mice
without the mutation were born resulting in a lot of effort put forth with a low return (Abbasi et al. 2018). Researchers
also attempted injection techniques. First, a single-guide RNA (sgRNA) and the Cas9 messenger RNA was injection into
the zygote’s cytoplasm. Thereafter a plasmid-based delivery system was soon developed where the plasmid vector
pX330, which expressed the sgRNA and Cas9, was injected into the fertilized eggs’ pronucleus (Abbasi et al. 2018). The
pronuclear injection method was also used in a different study where scientists were successful in stopping
spermatogenesis from occurring in male mice thus producing infertility (Bai et al. 2016). While both techniques resulted
in similar mutation rates, using the DNA-based pronuclear injection method was determined to be easier overall
(Abbasi et al. 2018). Finally, the electroporation method was utilized by shocking the cell with a high-pulse of electricity
to temporarily open a cell membrane pore so that a Cas9 protein–RNA complex could be incorporated (Abbasi et al.
2018). Regardless of technique, all four methods produced normally developed adult mice after being genetically
manipulated as embryos.
With such powerful gene-altering tools at hand, it is plausible that these can be applied to larger mammals like
cheetahs. Specifically, for this study, the pronuclear injection and electroporation methods would be the best utilized.
As seen in the previously mentioned spermatogenic cell study, CRISPR/Cas 9 is a powerful tool that can stop
reproduction from occurring (Bai et al. 2016). This means that it is might be possible to use the same pronuclear
injection method to help increase the fertility in cheetahs. The electroporation method was also found to produce
successful results with CRISPR/Cas9 as well. In comparison to the pronuclear injection technique, this method was
found to be highly successful on a large-scale genomic editing process that required less training, had a high survival
rate, and was highly efficient (Hashimoto and Takemoto 2015). Even though the electroporation method is generally
considered better than pronuclear injection, both techniques need to be explored regarding larger mammal
application.
CRISPR/Cas9 mechanisms have a wide range of advantages when used for genomic modification. It is far
cheaper, more efficient at cleaving DNA, and more user-friendly than other gene-editing tools like MNs, ZFNs, and
TALENS (Memia et al. 2018). This system also possesses tag insertion, double knock out, and point mutation capabilities
which further enhances its efficiency (Abbassi et al. 2018). The disadvantages of this gene-editing mechanism are
mainly ethical, but there have also been reports of off-target deletions and rearrangements with no one specific result
(Memia et al. 2018).
If this study were successful in producing viable cheetahs through either electroporation or pronuclear
injection, it would be revolutionary in application-based CRISPR research. Therefore, the ethical argument must be
considered as it would be irresponsible to ignore the fact that the next target organism may be humans consequently
putting CRISPR gene therapy, gene enhancement, and somatic or germ-line gene edits at the central focus of the
conversation. For example, gene therapy applied to somatic cells is generally accepted whereas germ-line cells are not
since the patient cannot pass on the mutation to their offspring unlike germ-line cells (Memia et al. 2018). Editing of
germ-line cells is burdened with three difficulties: the necessity of gene-editing, the unidentified risks of gene-editing,
and the hypothetical possibility of eugenics (Memia et al. 2018). These ethical concerns must be noted; however, these
concerns are steeped in fear of the unknown. This study will provide evidence through genetic modification
experimentation thereby giving clarity of the unknown.
Captive breeding, while proven successful in producing viable offspring and avoiding further loss of
heterozygosity within captive individuals, does not have the long-term ability to provide a stable population as there is
still a lack of genetic diversity within the wild (Terrell et al. 2016). While increasing this genetic diversity is possible
through gene-editing tools like TALENs and ZFNs, it is more efficient through the CRISPR/Cas9 mechanism. Therefore,
this study will determine if there is a correlation between genetic diversity and a stable population increase – wherein
fertility and mortality conditions remain relatively constant – through the large-scale application of electroporation and
pronuclear injection techniques (INED 2019).

III. Methods
Study Area
San Diego will act as the project’s home base as it is home to the San Diego Zoo. The San Diego Zoo will
be a resourceful potential partner as they have successfully produced 157 cheetahs since 1970 and are a part of
the Breeding Center Coalition (BCC) which works towards the goal of a sustainable cheetah population (San
Diego Zoo 2019). The BCC is a group of nine accredited zoos and aquariums that freely share information on
the best practices of captive breeding of cheetahs in North America (Cincinnati Zoo 2016). One technique the
BCC has attempted for multiple years is artificial insemination, however they have yet to produce a successful
pregnancy or viable offspring (National Zoo 2018). By partnering with the San Diego Zoo, we will have access to
this and other information that can be utilized for further research or to fine tune our current study. The San
Diego Zoo will also be where we will rear our captive group since they already have a large off-exhibit cheetah
enclosure as well as a Veterinary Medical Center (San Diego Zoo 2019).
Botswana’s Mokolodi Nature Reserve, Chobe National Park, and Mashatu Game Reserve will also act as
study areas. The most recent estimate of cheetahs was done in 2002 by the Department of Wildlife and Parks
(DWNP); it was determined that there were 1,768 wild cheetahs left in Botswana (Majelantle 2005). While
there has been no recent estimation on Botswana’s wild cheetah population, there are roughly 4,190 adults in
Southern Africa which includes Botswana (IUCN 2015). It has also been previously determined that Botswana
cheetah populations have a wider range of genetic diversity which could further help stabilize the population
when the genetically modified individuals have been released (Dalton et al. 2013). The Mashatu Game Reserve
and Chobe National Park have diverse landscapes from grasslands and open plains to marshes which would be
beneficial in observing cheetah in multiple ecosystems (Chobe National Park 2019, Mashatu 2019). These two
reserves will also allow the cheetahs adequate room to live and interact with Mashatu covering about 29,000
hectares while Chobe boasts an impressive 154,000 hectares (Mashatu 2019, Breathe Travel 2016). Both
reserves will be used to monitor and observe individuals of group A and will eventually be where group D is
released. The Mokolodi Nature Reserve will be where group C is released within an enclosed area to monitor
the individuals’ interactions with the wild. Since Mokolodi is only 3,700 hectares, this will be the best
reservation to monitor the more isolated group of cheetahs (Mokolodi Nature Reserve 2019).

Experimental Design
The study will be conducted year-round and it will take around 30 years to complete for two reasons.
First, the lifespan of a captive cheetah is about fifteen years which will account for two generations of
individuals reared in captivity (Big Cat Rescue 2016).
Second, more accurate data can be analyzed when
monitoring multiple generations amongst all groups.
Group A is our control group. These individuals will
be collared, wild cheetahs within the Mashatu
Game Reserve and Chobe National Park that we will
observe in their natural environment. Group B will be
genetically engineered individuals that are reared in
captivity. The parents will be obtained through the San
Diego Zoo and will have a sex ratio of seven males to seven
females. Female cheetahs produce an average of about
three cubs per litter which would allow for a minimum of
twenty-one embryos to genetically modify of which ten
female and ten male cubs will be chosen to be a part of
group B (Marker 2002). If group B is successful, group C will
Fig. 1: Arrows correspond to the offspring of the
consist of the offspring of group B. Group C will eventually
previous group. The individuals of groups B and
be released back into the wild within the Mokolodi Nature C will be directly partitioned via physical barriers
so that interactions will be limited to individuals
Reserve, kept as separate as possible from wild cheetahs,
that share the two deviations of the
and their interactions with their environment (i.e. CRISPR/Cas9 technique. Group D individuals will
be kept geographically separate as they will be
environmental factors, hunting and mating capabilities,
in different reservations entirely.
etc.) will be monitored through telemetry collars and check-
ups. This will be done so that it can be determined if the group can survive without human influence. If group C
is successful in producing viable offspring, group D will consist of these offspring that will be released and
allowed to commingle with group A. The offspring of groups D and A will be the focus of this study and their
success rate will be based off the diversity of their genes, fertility rate, lifespan, and susceptibility to diseases.
Our sample size will consist of roughly 184 individuals. Group A will consist of twenty individuals that
will not be directly manipulated. Group B will also have twenty individuals. Group C will have thirty individuals,
while group D will have forty-five individuals that will produce roughly sixty-nine offspring.
In group B, five females and five males that have been genetically modified through the electroporation
method will be chosen once the females have reached sexual maturity at two years of age, and bred together
to ideally produce a minimum of fifteen cubs (TOLWEB 2008). This will be repeated with another five females
and five males that have been genetically modified through the pronuclear injection method. Around thirty
cubs will be produced by group B and these individuals will now be considered group C. The same procedure
will be conducted with group C wherein roughly fifteen cheetahs that are a result of either electroporation or
pronuclear injection will be bred together for an ideal minimum of forty-five cubs. Group D will consist of these
forty-five individuals and the same procedure will again be conducted to yield another ideal minimum of about
sixty-nine cubs. Roughly half of group D’s offspring will have been the result of the electroporation technique
and will be released within the Mashatu Game Reserve while the individuals produced through pronuclear
injection will be released within Chobe National Park to interact with group A.
Any direct offspring these individuals produce will be closely monitored, but not directly manipulated
as groups B – D were. This is so the natural progression of the population can be observed without human
influence. This experimental design is illustrated in Fig. 1, along with additional methodology information on
how the individuals produced by the two different techniques will be kept separate.
Any offspring past the second generation of each group (i.e. grandchildren) will be collared, but not as
intensely monitored. This is because if healthy individuals are produced in the third generation and can leave
their mother, this project will be considered successful and further intense study is unnecessary. However,
continued observation is recommended so that further changes – whether genetic, phenotypic, or otherwise –
can be noted.
For the control group, we will measure population density, average lifespan, determine the home
ranges of individuals, the amount of genetic homozygosity, and amount of offspring produced. The same
factors will be measured in groups B, C, and D. However, because group B is studied within captivity there will
be no home range calculated. Additionally, susceptibility to disease, fertility, and increased genetic
heterozygosity will be studied in groups B – D and determine if these factors have any correlation to survival.
Those in captivity will be monitored monthly (in addition to daily upkeep), while those in the wild will be
monitored roughly every three to four months so there is as little human interference as possible.

Research Methods
 Genetic modification will be applied to the embryos using the two CRISPR/Cas9 techniques mentioned
previously: electroporation and pronuclear injection. These two techniques will target and delete five
homozygotic mutations within the gene AKAP4. These mutations have been thought to be the primary reason
for the high amounts of damaged sperm within cheetahs (Dobrynin et al. 2015). With these deletions, it is
thought that a higher fertility rate will be induced.
The genomes of group B’s parents will be sequenced by Medical Neurogenetics Laboratories to
determine the most heterozygotic genes (MNG Labs 2019). Either by electroporation or pronuclear injection
these gene sequences will then be targeted and integrated within the zygotes.
Using a tranquilizer gun (Pneu-Dart’s Model 389 Projector) and a drug cocktail of 50μg/Kg
Medetomidine and 5mg/Kg of Ketamine, individuals not contained within the San Diego Zoo will be sedated
from a distance so that blood and urine samples can be taken (Pneu-Dart; Mwangi et al. 2016). Blood samples
will help us determine presence of diseases, red and white blood cell count, and genetic diversity that will be
measured through DNA sequencing and used to determine the frequency of heterozygosity. These blood
samples will be sent to the Laboratory Corporation of America for analysis (LabCorp 2019). Urine samples will
help determine general health of the individual by monitoring dehydration and other diseases. The same
samples will be taken from the San Diego Zoo individuals, but any sedative and tranquilizing equipment should
be provided by the San Diego Zoo.

Data Analysis
Cohort life tables will be constructed for each group to determine mortality rates, stage-specific
fecundity, and reproductive rates. This is going to be our most important collection of data since these would
be groundbreaking techniques and any factor affected would need close consideration. Genome maps and
pedigree charts will be used to keep track of individuals produced. We will compare these samples across all
groups to determine if heterozygosity is in fact developing. Finally, GIS analysis will be required to analyze
geographic distances of the individuals’ home ranges. We will present our findings at the Fall Expo of the
Wildlife Conservation Network in 2050 (WCN 2019).

IV. Expected Contribution

If our study is successful it will help zoos, wildlife reservations, and preserves to better manage their
cheetah populations. If we’re able to prove there is a correlation between increased genetic diversity and
increased fitness, breeding programs could be implemented as well. However, the success of this study is not
limited to just cheetah populations as it could also be applied to other species populations such as the critically
endangered northern and southern white rhinoceros (Ceratotherium cottoni and Ceratotherium simum
respectively), the Bornean orangutan (Pongo pygmaeus), and the endangered chimpanzee (Pan troglodytes)
 (Cinková and Bičík 2013; WWF 2019). Ultimately, this technology has never been attempted on wild
populations outside of laboratory control and, if successful, can be utilized to increase multiple species
populations.
The ethics of this experiment must be stressed because if successful, the possibility that these
treatments could be used on human subjects is high. Where is the line drawn between necessary genetic
editing and eugenics? What species are worth saving and how do we determine that? These are imperative
questions that need to be debated thoroughly with both the public and the scientific community. However,
while gene editing mechanisms are highly controversial, the reward outweighs the risk. If animals that are on
the verge of extinction, like the cheetah, can be helped or even saved through these gene editing mechanisms,
they should be attempted.

V. Schedule of Activities
A. Groups A, C, and D: check in every 3-4 months
1. Mashatu Reserve, Chobe National Park, and Mokolodi Nature Reserve
a. Days 1 – 14: determine location of the individuals that have been tracked for roughly three
months and determine the size of their home range through GIS. Collect blood and urine
samples.
b. Day 15: send data to lab for analysis and return San Diego. Allow 1 – 6 weeks for analysis
completion.
c. Day 16: after receiving lab data, fill in life tables, genome maps, and pedigree charts. Make
note of any concerning factors that could be due to genetic modification such as debilitating
diseases.
B. Group B: check in every month
1. San Diego Zoo
a. Days 1 – 5: collect blood and urine samples.
b. Day 6: send data to lab for analysis. Allow 1 – 6 weeks for analysis completion.
c. Day 7: after receiving lab data, fill in life tables, genome maps, and pedigree charts. Make
note of any concerning factors that could be due to genetic modification such as debilitating
diseases.
C. Final Data Analysis
1. Collect Final Data Analysis
a. Days 1 – 14: compile all previous individual data into one pedigree chart, conduct final
statistical analysis, compare any outliers, understand major trends.
 b. Days 15 – 30: publish findings, share final information with our partners, and make a
presentation describing our findings.
2. Wildlife Conservation Network
a. Days 1 – 2: present findings at the 2050 Fall Expo, national WCN in San Francisco, California.

VI. Budget
$$ Links
Salary/Wages
Project Manager #1 $70K/yr https://www.glassdoor.com/Salaries/research-manager-salary-
SRCH_KO0,16.htm
Project Manager #2 $70K/yr https://www.glassdoor.com/Salaries/research-manager-salary-
SRCH_KO0,16.htm
Veterinarian $85K/yr https://www.glassdoor.com/Salaries/veterinarian-salary-
SRCH_KO0,12.htm
Vet Technician $11/hr (x 8 hrs x 14 https://wfscjobs.tamu.edu/jobs/veterinary-technician-or-assistant-
days) =$1,232 wisconsin/
GIS Technician $16 - 18/hr (x 8 hrs x 14 https://wfscjobs.tamu.edu/jobs/ops-biological-scientist-ii-florida/
days) = ~$1,904
Intern #1 $0 https://wfscjobs.tamu.edu/jobs/cheetah-conservation-program-
south-africa-4/
Intern #2 $0 https://wfscjobs.tamu.edu/jobs/cheetah-conservation-program-
south-africa-4/
Total $228,136

Equipment
CRISPR/Cas9 Use $65 https://stanmed.stanford.edu/2018winter/CRISPR-for-gene-editing-
is-revolutionary-but-it-comes-with-risks.html
Garmin T 5 250 per collar 164 https://www.uglydoghunting.com/shop/for-your-dog/training-
Additional Colar individuals = $41,000 collars-gear/additional-t5-tracking-dog-collar/?gclid=Cj0KCQiAk-
7jBRD9ARIsAEy8mh7MxST-2mV9W20TFUYtdSO4mvgR7FoL_BKkHS-
RqVqjdZfbydoWVngaAjUUEALw_wcB
Medetomidine 9,200μg (~9.2mg) = $80 https://www.medchemexpress.com/medetomidine.html
Ketamine 920mg = ~$212 https://www.boundtree.com/Pharmaceuticals/Class-IV-
Drugs/Ketamine%2C-Class-III/p/group002326
Tranquilizer Gun $976.45 https://shop.pneudart.com/animal-control-389-long-range-
package/
Darts $13.75 x (164 https://shop.pneudart.com/1.5cc-type-c-rdd/
individuals) = $2,255
Truck $15,998 https://www.carmax.com/cars/ford/ranger
Enclosure $443,520 https://www.homedepot.com/p/YARDGARD-6-ft-x-50-ft-9-Gauge-
Equipment Galvanized-Steel-Chain-Link-Fabric-308806A/202024344
Lab Analysis $50/hr (x minimum 40 https://www.upwork.com/hiring/data/how-much-hire-data-
(Estimation) hrs) = $2000 scientist/
Genome Mapping $1,400 x (164 https://www.cnbc.com/2015/12/10/unlocking-my-genome-
Analysis individuals) = $229,600 was-it-worth-it.html
 Total $734,730.45

Vaccinations (x 7)
Hepatitis A $113.99/dose, 2 dose https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
series ($1,595.86)
Hepatitis B $89.99/dose, 3 dose https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
series ($1,889.79)
Typhoid $142/adult ($994) https://www.cvs.com/minuteclinic/services/price-lists
Yellow Fever $110/adult ($770) https://sightdoing.net/travel-vaccines-cost/
MMR $99.99/dose ($699.93) https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
TDAP $63.99/dose ($447.93) https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
Chickenpox $149.99/dose, 2 dose https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
series ($2,099.86)
Pneumonia $125.99/dose ($881.93) https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
Influenza $40.99/dose ($286.93) https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
Meningitis $133.99/dose ($937.93) https://www.walgreens.com/topic/healthcare-clinic/price-menu.jsp
Polio $115/adult ($805) https://www.cvs.com/minuteclinic/services/price-lists
Total $11,409

Travel
Peermont Mondior $109/night (x 14 nights) https://www.tripadvisor.com/
Gaborone = $1,526
Botswana flight per $1,580 x 7 https://www.safaribookings.com/botswana/getting-there
person
Conference Fees $50 per person x7 https://donate.wildnet.org/2019SpringExpo?ms=eventsbutton_se19
Hotel: 3 night stay $94 per person x 7 https://www.google.com/travel/hotels/
in San Fran
San Fran flight from Flight $89 per person x7 https://www.delta.com/flight-search/search-
San Diego results?cacheKeySuffix=27aca9df-a60b-483d-8335-4e0d49ac6e37
Total $14,217

Grand Total
$988,493

*Information on possible vaccines for traveling to Botswana can be found here:

https://www.passporthealthusa.com/destination-advice/botswana/.

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