DOI: 10.1111/risa.

13948

ORIGINAL ARTICLE

Bayesian network-based risk assessment of synthetic biology:

Simulating CRISPR-Cas9 gene drive dynamics in invasive rodent
management

Ethan A. Brown Steven R. Eikenbary Wayne G. Landis

Institute of Environmental Toxicology and Abstract

Chemistry, College of the Environment, Western
Washington University, Bellingham, Washington,
USA
Correspondence
Ethan A. Brown, Biological Sciences Department,
University of Notre Dame, Notre Dame, IN, USA.
Email: ebrown23@nd.edu
Gene drive technology has been proposed to control invasive rodent populations as
an alternative to rodenticides. However, this approach has not undergone risk assess-
ment that meets criteria established by Gene Drives on the Horizon, a 2016 report by
the National Academies of Sciences, Engineering, and Medicine. To conduct a risk
assessment of gene drives, we employed the Bayesian network–relative risk model to
calculate the risk of mouse eradication on Southeast Farallon Island using a CRISPR-
Cas9 homing gene drive construct. We modified and implemented the R-based model
“MGDrivE” to simulate and compare 60 management strategies for gene drive rodent
management. These scenarios spanned four gene drive mouse release schemes, three
gene drive homing rates, three levels of supplemental rodenticide dose, and two timings
of rodenticide application relative to gene drive release. Simulation results showed that
applying a supplemental rodenticide simultaneously with gene drive mouse deploy-
ment resulted in faster eradication of the island mouse population. Gene drive homing
rate had the highest influence on the overall probability of successful eradication, as
increased gene drive accuracy reduces the likelihood of mice developing resistance to
the CRISPR-Cas9 homing mechanism.

KEYWORDS
Bayesian network, gene drive, integrated pest management, risk assessment, synthetic biology

1 INTRODUCTION manner, resulting in probabilistic estimates of gene drive

Proposed environmental applications of synthetic biology
bring with them a host of novel management and regula-
tory considerations. Chapter 6 of Gene Drives on the Horizon
(National Academies of Sciences, Engineering, & Medicine
[NASEM], 2016) and Landis et al. (2020) describe frame-
works for risk assessment of synthetic biology using the
Bayesian network–relative risk model (BN-RRM), with gene
drives as an example of a synthetic biology-derived stres-
sor. Using the BN-RRM framework, we constructed a prob-
abilistic risk assessment model for the theoretical intro-
duction of gene drive modified (GDM) house mice (Mus
musculus) to suppress the invasive mouse population on
Southeast Farallon Island (SEFI) and protect endangered,
indigenous seabirds such as the ashy storm petrel (Ocean-
odroma homochroa). This work demonstrates that gene
drive risk assessment can be conducted in a quantitative
efficacy.
1.1 Introduction to synthetic biology
The National Academies of Sciences, Engineering, and
Medicine (NASEM) define synthetic biology as the genera-
tion of novel traits or organisms via the implementation of
chemically synthesized or computationally designed DNA
that does not occur in nature. One branch of synthetic biology
is genome editing, defined as the introduction of new alleles
or genes into an organism (NASEM, 2016). Clustered reg-
ularly interspaced short palindromic repeats (CRISPR) Cas
(CRISPR-Associated) systems are a common tool in genome
editing. CRISPR-Cas9 systems can modify specific DNA
sequences using a complementary guide-RNA (gRNA) com-
plexed with a DNA-targeting endonuclease (Cas9) that cre-

Risk Analysis. 2022;42:2835–2846. wileyonlinelibrary.com/journal/risa © 2022 Society for Risk Analysis. 2835
2836
ates a double-strand break at a target DNA sequence in an
organism’s genome. We use the term “homing rate” to mean
the probability of the CRISPR-Cas9 complex successfully
locating the target DNA sequence and then “cutting rate” to
mean the probability of the Cas9 endonuclease successfully
causing a double-strand break.
After the CRISPR-Cas9 complex successfully locates and
cuts the target DNA sequence, the site of the double-strand
break is then repaired by either homology-directed repair
(HDR), where the cut DNA sequence is successfully replaced
by the desired DNA sequence (payload gene), or by nonho-
mologous end joining (NHEJ), where small frameshift muta-
tions occur, leading to incorrect insertion of the payload gene
(Knott & Doudna, 2018). The level of gene editing precision
offered by CRISPR-Cas systems has the potential to alter the
germ lines of populations. Genome editing for the purpose of
altering populations is the conceptual foundation behind gene
drive technology (NASEM, 2016).
1.2 Gene drives and their environmental
applications
Gene drives are naturally occurring or engineered genetic
constructs that transfer to offspring at proportions greater
than the Mendelian expectation (i.e., above 50%) (NASEM,
2016). CRISPR-Cas9-based gene drives have been synthe-
sized (Hammond et al., 2016) to “drive” engineered genes
through target populations. First, the CRISPR-Cas9 con-
struct “cuts” the wild-type allele. Then, the wild-type gene is
replaced by a new (payload) gene via HDR. Finally, the pay-
load gene is inserted and replicated throughout the genome,
making the organism homozygous for the payload allele
(Webber et al., 2015).
Intended applications of gene drives fall into two broad cat-
egories: population replacement and population suppression.
The goal of population replacement is to substitute a target
wild-type gene with the desired payload gene, avoiding a sub-
stantial fitness reduction that could result in a decline of the
target population. The goal of population suppression is to
reduce or eliminate the target population by substituting the
target wild-type gene with a gene that reduces organismal
fitness (Marshall & Akbari, 2016). Gene drives have been
proposed for controlling vector-borne diseases, suppressing
invasive species, and inducing pesticide tolerance or pesticide
sensitivity in agriculture (NASEM, 2016).
1.3 Controversies and uncertainties
surrounding gene drive use
Gene drives have come under scrutiny, with researchers not-
ing their potential for adverse environmental impacts and
questioning their ability to successfully suppress or spread
desired traits through populations. Webber et al. (2015) sug-
gests the possibility of GDM organism dispersal into nontar-
get populations, leading to unintended extinctions and alter-
BROWN ET AL.
ations to community dynamics. Roberts et al. (2017) has
postulated that hybridization of GDM organisms with sim-
ilar species in a region could spread gene drives to nontar-
get species, leading to unintended population suppression or
replacement.
The development of organismal resistance to the hom-
ing properties of gene drives has also been investigated.
Resistance occurs when frameshift mutations alter the DNA
sequence being targeted by the CRISPR-Cas9 complex, ren-
dering the sequence unrecognizable and preventing hom-
ing and cutting of the target gene. These mutations can
occur when the Cas9 double-strand break is repaired by
NHEJ instead of HDR, or when HDR incorrectly or incom-
pletely inserts the payload gene (Marshall et al., 2017). In
this study, we will reference two types of resistance gener-
ation: in-frame and out-of-frame resistance. In-frame resis-
tance refers to a mutation that results in zero fitness reduc-
tion to organisms, essentially creating a new genotype that
is unrecognizable by the CRISPR homing mechanism and
also carries no fitness cost. Out-of-frame resistance refers to
mutations that disrupt gene functioning and result in nonvi-
able organisms. Development of in-frame resistance has the
potential to render gene drives obsolete in targeted popu-
lations by creating a germline that is completely indepen-
dent of the CRISPR-Cas9-enhanced inheritance mechanism
(Sánchez et al., 2020). Development of in-frame and out-of-
frame resistance has been demonstrated in laboratory test-
ing of a CRISPR-Cas9 gene drives targeting Drosophila
melanogaster (Champer et al., 2017; Kandul et al., 2020) and
Aedes aegypti (Li et al., 2020).
To overcome potential gene drive resistance, studies have
proposed using a multiplexing approach to create gene drive
constructs that target multiple DNA sequences in a gene using
multiple gRNA sequences. This approach is expected to min-
imize the probability of resistance development by increasing
the number of mutations required to negate the homing capa-
bilities of CRISPR-Cas9 gene drives (Marshall et al., 2017;
Noble et al., 2017, 2019). Champer et al. (2020) tested a mul-
tiplexed CRISPR-Cas9 gene drive that successfully spread to
fixation through a laboratory population of D. melanogaster.
The drive utilized two gRNAs, each targeting a haplolethal
gene. Haplolethal genes are essential to organism function-
ality, meaning mutations are less likely to cause in-frame
resistance. While many multiplexed gene drive constructs
have been proposed, few have undergone laboratory testing
(Champer et al., 2020; Kandul et al., 2020; Li et al., 2020;
Wolabu et al., 2020), and fewer have demonstrated an abil-
ity to negate the development of resistance (Champer et al.,
2020). As of October 2021, no laboratory CRISPR-Cas9 gene
drive studies have been conducted using M. musculus.
1.4 Why risk assessment?
Risk assessment is a causal and empirical process that
is well-suited for calculating probabilistic estimates of
synthetic biology-related impacts to ecological systems
BAYESIAN NETWORK GENE DRIVE RISK ASSESSMENT
(Landis, Brown et al., 2020; NASEM, 2016). Risk assess-
ment is defined as the calculation of risk for one or more end-
points due to the effects of one or more stressors, where risk
is defined as the entire probability distribution of potential
impacts to a management-defined endpoint (NASEM, 2016).
Gene Drives on the Horizon (NASEM, 2016) recommends
the use of the BN-RRM to conduct risk assessment of syn-
thetic biology. The BN-RRM utilizes a conceptual modeling
approach to relate site-specific variables and potential man-
agement strategies into a causal framework relating sources
of ecological stressors to endpoints of concern to managers or
regulators. Bayesian networks (BNs) are then used to calcu-
late estimates of risk by relating variables through conditional
probability (Landis, Brown, et al., 2020; Landis et al., 2017;
Landis, Chu, et al., 2020; Landis, 2021). The BN-RRM is a
useful framework for combining advanced ecological model-
ing techniques into an intuitive tool for adaptive management
(Landis et al., 2017).
1.5 Introduction to Bayesian networks
BNs are acyclic, probabilistic models that relate a network
of variables through conditional probability. Relationships
between nodes are separated into two primary classifications:
parent nodes and child nodes (Marcot et al., 2006). Arrows in
a BN represent causal linkages between variables, with parent
nodes influencing child nodes (Landis, Brown, et al., 2020;
Landis, Chu, et al., 2020). Child nodes are derived by condi-
tional probability tables (CPTs) that calculate the probabilis-
tic relationship between a child node and its parent nodes.
Nodes within a BN contain multiple variable states and CPTs
contain the probabilities of each child node’s variable states
given all possible combinations of parent node states. Nodes
can be both children and parents depending on their location
within the network structure (Marcot et al., 2006; McDonald
et al., 2015).
Sensitivity analyses can be run in a BN to calculate the
mutual information between parent and child nodes. Mutual
information is the degree of shared information between pairs
of parent and child nodes and is used in BNs to calculate the
relative entropy reduction of parent nodes on a child node,
estimating the degree of influence each parent variable exerts
on a child variable (Hosack et al., 2008; Marcot, 2012). Sen-
sitivity analysis is a powerful tool for identifying knowledge
gaps in a BN-RRM risk assessment, in addition to quantify-
ing the amount of variance explained by each set of nodes.
1.6 Case study: Southeast Farallon island
The goal of this work was to demonstrate the use of the
BN-RRM in a risk assessment of synthetic biology that con-
forms to the criteria established by Gene Drives on the Hori-
zon (NASEM, 2016). These criteria include (1) quantitatively
tracing causal pathways outlined in the conceptual model
2837
for the risk assessment scenario, incorporating the state-of-
the-knowledge on causal interactions; (2) calculating risk
to management-defined endpoints, incorporating site-specific
variables and the concerns of relevant managers, stakehold-
ers, and publics; (3) elucidating sources of uncertainty and
weighing the relative influence of parameters on risk; and
(4) allowing for comparison between multiple management
strategies with the ability to weigh costs and benefits in the
context of multiple endpoints.
We used SEFI as a case study for gene drive risk assess-
ment, demonstrating the ability of the BN-RRM and simi-
lar approaches to meet NASEM criteria. SEFI is populated
by thousands of invasive M. musculus which pose a hazard
to seabirds of conservation concern. Specifically, the high
density of M. musculus on SEFI attracts the migratory bur-
rowing owl (Athene cunicularia). Once the owls deplete the
mouse population, they begin to feed on ashy storm petrels
(O. homochroa), an endangered seabird of which half the
global nesting population resides on the Farallones (Inter-
national Union for Conservation of Nature [IUCN], 2018;
United States Fish & Wildlife Service [USFWS], 2019a). We
modified and used the R-based model “MGDrivE” (Sánchez,
Wu et al., 2020; Sánchez, Bennett et al., 2020) to compare
mouse eradication risk between 60 simulated management
scenarios, varying gene drive homing rate, as well as, the tim-
ing and dosage of supplemental rodenticide use. The specific
gene drive modeled in simulations was the Sox9 CRISPR-
Cas9 gene drive, which is intended to spread female (XX)
sterility through mouse populations through the release of
modified males (XY). XX mice who inherit the Sox9 gene are
hypothesized to be completely sterile, in addition to develop-
ing male morphology (Prowse et al., 2017). We selected the
Sox9 construct for our simulations because it has been a major
topic of discussion as an emerging tool for mouse popula-
tion suppression (Campbell et al., 2019; Prowse et al., 2017).
Additionally, there is empirical evidence that Sox9 expression
in XX mice leads to development of male morphology (Vidal
et al., 2001), making the Sox9 CRISPR-Cas9 gene drive a
viable candidate for future laboratory studies.
2 METHODS
First, we created a conceptual model outlining sources-to-
endpoint causal pathways for mouse eradication on SEFI
(Figure 1). Next, we created a BN (Figure 2) with nodes for
gene drive homing rate, gene drive mouse release scheme,
rodenticide dose, and rodenticide timing. Then, we used the
modified version of MGDrivE to calculate the probabili-
ties of mouse eradication for 60 unique management sce-
narios, parameterizing the BN with the simulation outputs.
Finally, we conducted sensitivity analysis on the parame-
terized BN to estimate the relative influence of gene drive
homing rate, rodenticide dose, and timing of the roden-
ticide application on the probability of successful mouse
eradication.
2838 BROWN ET AL.

F I G U R E 1 Conceptual model for the SEFI invasive M. musculus case study, constructed using the framework of the Relative Risk Model, with sources
releasing stressors that persist in habitats, causing effects, and eventually impacts to management endpoints. The two sources in this case study are the
deployment of Sox9 gene drive mice and brodifacoum rodenticide baits. The stressors are exposure to brodifacoum and gene drive variables such as homing
rate and the probability of in-frame resistance generation. Effects from these stressors are altered heredity due to the CRISPR gene drive, interactions of
stressors with different life-stages of mice, toxicity-induced mortality from brodifacoum, changes to overall abundance of mice, influence of patch dynamics
on eradication, effects of the gene drive on mouse mating patterns, and temporal trends in population dynamics. The endpoint is the probability of mouse
eradication on SEFI, where the two possible outcomes are successful or unsuccessful eradication. Other sources and stressors not evaluated in our models are
listed as assumptions under the sources-to-stressors diagram. We list impacts to burrowing owl and ashy storm petrel populations as potential endpoints of
management concern, however, we did not quantify risk to these populations in our simulations because of our focus on parameterizing the gene drive
pathway. Examples of how to quantify risk of impacts to non-target species due to species interactions and toxicity can be found in Landis et al. (2017) and
Landis, Chu et al. (2020)

2.1 Management scenarios
Integrated pest management (IPM) is a strategy employed
in pest eradications that focuses on minimizing the use of
chemical pesticides to prevent effects to nontarget species.
The purpose of IPM is to alter the risk of nontarget impacts
by decreasing the amount of pesticide used, or by employ-
ing alternate methods to reduce the probability of nontarget
species toxicity from pesticides. The United States Fish and
Wildlife Service (USFWS) released an Environmental Impact
Statement (EIS) (USFWS, 2019a) recommending the use of
the rodenticide brodifacoum to eradicate mice on SEFI. Brod-
ifacoum is an anticoagulant that functions through vitamin K
antagonism. The EIS also recommended a number of IPM
strategies for minimizing seabird exposure to brodifacoum.
Methods included using hazing tactics to frighten western
gulls (Larus occidentalis) away from SEFI during the roden-
ticide deployment period and timing rodenticide deployment
to coincide with the annual low point of the mouse population
(USFWS, 2019a).
Gene drives have been proposed as a method of IPM that
would serve as a biological control agent for eradicating pest
populations, potentially reducing or eliminating the need for
pesticide use (NASEM, 2016; Webber et al., 2015). We sim-
ulated gene drive scenarios across four GDM mouse release
schemes (Table S1), three levels of gene drive homing rate,
three doses of brodifacoum (including zero brodifacoum),
and two timings of brodifacoum deployment, for a total of 60
scenarios, each simulated for 1000 Monte Carlo repetitions.
See Table S2 for a complete list of simulated scenarios.
2.2 Analysis tools
To calculate the influence of GDM M. musculus and sup-
plemental brodifacoum use on the risk of SEFI mouse erad-
ication, we implemented three numerical models. First, we
applied exposure-response analysis using the “drc” package
in R (Ritz & Streibig, 2020) to model the probability of M.
musculus mortality given a specified dosage of brodifacoum.
Next, we constructed a BN using Netica™ (Norsys Software,
Corp.). Finally, we modified and used MGDrivE (Sánchez,
Bennett et al., 2020) to simulate GDM mouse population
dynamics and population genetics for each management sce-
nario listed in Table S2.
2.2.1 Brodifacoum exposure-response
modeling using R-‘drc’
Using the “drc” package in R (Ritz & Streibig, 2020) we
generated a dose–response equation relating dosage of brod-
ifacoum with M. musculus mortality. To generate the dose–
response curve (Figure S1) and equation (Equation 1), we fit-
ted a two-parameter log-logistic binomial regression model
using toxicity data from Wheeler et al. (2019). We calcu-
BAYESIAN NETWORK GENE DRIVE RISK ASSESSMENT 2839

F I G U R E 2 BN for evaluating the mouse eradication endpoint for SEFI. Nodes for time, pesticide dose, pesticide timing, homing rate, and GDM mouse
release scheme feed into a node describing mouse abundance. Node states for mouse abundance include “Zero” (zero mice), “Sparse” (1–1000 mice), “Low”
(1001–10,000 mice), “Med” (10,001–25,000 mice), and “High” (25,001–50,000) mice. BN is currently showing MGDrivE simulation results for
Management Scenarios 1 and 9 (Table SII) at year 7 following the initial deployment of GDM mice. Scenario 1 assumes a low homing rate and no
supplemental rodenticide, leading to a low (3.7%) probability of eradication after 7 years. Scenario 9 assumes a high homing rate and a large dose of
supplemental rodenticide, resulting in a high (90.5%) probability of eradication after 7 years. The gray nodes represent the burrowing owl and ashy storm
petrel endpoints, which were not evaluated in MGDrivE simulations, as our focus was on quantifying the efficacy of the Sox9 gene drive using the BN-RRM

lated 95% prediction intervals and 95% confidence intervals
to show the degree of confidence in point estimates along the
curve. Daily mortality probability was extrapolated from the
7-day mortality curve using Equation 2.
1 com/netica.html.
P(7day Mortality|Dose) =
1 + exp (−3.30 (log (dose) − log 1.76))
P(7day Mortality|Dose)
P (Daily Mortality) =
7
2.2.2 Bayesian network model structure
We created the BN for SEFI mouse eradication using Net-
ica™. BNs constructed in Netica can be viewed by down-
loading the free version of Netica from http://www.norsys.
The BN (Figure 2) contains nodes for gene drive, pesti-
(1)
cide, and time variables. The node for brodifacoum dose (pes-
ticide dose) assumes a constant level of pesticide exposure
(in mg/kg) over an 8-week simulated exposure period. The
(2) selected brodifacoum doses were 0, 1.26, and 2.02 mg/kg,
2840
which correspond to 7-day LD0 , LD25 , and LD60 values,
respectively. Next, the node for brodifacoum deployment tim-
ing (pesticide timing) indicates whether brodifacoum was
deployed simultaneously with the first GDM mouse release
(Start) or after the final GDM mouse release (End). The
correct homing rate node contains states for 95% homing
rate, which corresponds to observed resistance generation
observed in laboratory testing of CRISPR-Cas9 gene drives
(Hammond et al., 2016); 99% homing rate, and 99.9% hom-
ing rate. Note that 99% and 99.9% homing rates have been
hypothesized for multiplexed gene drives (Marshall et al.,
2017). The GD Release Scheme node delineates four unique
release patterns of GDM mice (Table S1).
2.3 MGDrivE simulations
MGDrivE combines population genetics, age-class popu-
lation dynamics, patch dynamics, and flexible gene drive
parameters into an R-based model. Additionally, MGDrivE
provides options for altering biological parameters of
mosquitoes to accommodate a range of species and allows
for the adjustment of the number of ecological patches and
the probabilities of migration between patches. MGDrivE can
be run as a deterministic or stochastic model, with the abil-
ity to specify how many Monte Carlo repetitions to perform
(Sánchez, Wu et al., 2020). We ran MGDrivE as a probabilis-
tic model, running 1000 Monte Carlo repetitions for each sce-
nario. These 1000 stochastic repetitions were used to calcu-
late the CPTs for mouse abundance and mouse eradication
nodes in the BN, providing probabilistic estimates of the effi-
cacy of each management scenario across time.
We made several modifications to the MGDrivE source
code to accommodate M. musculus life cycle and population
dynamics, Sox9 gene drive functionality, toxicological effects
from a pesticide, and transformation of MGDrivE output into
CPTs for the BN.
2.3.1 Modifications to MGDrive
(MosquitoGD to MouseGD)
To alter the functionality of MGDrivE for compatibility
with modeling population dynamics of M. musculus, we
made changes to functions affecting mating behavior, den-
sity dependent mortality, and recruitment of early life stage
mice. We also added functions for modeling the dynamics
of the Sox9 CRISPR-Cas9 gene drive, including rodenticide-
induced mortality in simulations, and extracting the numer-
ical output of simulations into CPTs for BN parameteri-
zation. We modified the source code of MGDrivE version
1.5.0 (Sánchez, Bennett et al., 2020). This altered version of
MGDrivE can be found at https://github.com/eabrown2378/
MGDrivE. Table S5 contains descriptions of all parameters
used in the following equations which we modified from the
original MGDrivE package.
BROWN ET AL.
In the original version of MGDrivE by Sánchez
et al. (Sánchez, Bennett et al., 2020) (hereafter termed
“MosquitoGD”), all female mosquitoes are assumed to find
a mate and oviposit eggs once each day, assuming pan-
mixia unless genotype-specific mating preferences are spec-
ified by the user. In the altered version of MGDrivE (here-
after termed “MouseGD”), not all female mice mate and con-
ceive offspring every day. Instead, each female has a proba-
bility of mating and conceiving new offspring calculated by
Equation 3:
L
P (C) = (3)
365 days
where G is the number of newly gestating mice resulting from
a day of mating and Q is the average number of mouse pups
per litter. In parallel with the work of Prowse et al. (2017), we
assumed an average of six mouse pups per litter. The aver-
age number of pups (Q) was used as the rate parameter for a
Poisson distribution, calculating G from the summation of F
draws from the distribution.
In MosquitoGD age-class population dynamics, four
mosquito life stages were assumed: egg, pupa, larvae, and
adult, where density-independent mortality occurred at all
four life stages and density-dependent mortality occurred at
the larval stage (Sánchez, Wu et al., 2020). In MouseGD, we
also assumed four life stages: gestating, nursing, adolescent,
and adult, where density-dependent mortality is applied at the
adult life stage using Equation 6:
( )1
K ∗ 1∕2 𝜃
D= (6)
K + Ad
where L is the assumed average annual number of litters for
female M. musculus and P(C) is the daily probability of a
female mouse becoming pregnant. We assumed an annual
average of 7.5 litters in all MouseGD simulations. The num-
ber of female mice who mate and become pregnant during a
single day of MouseGD population dynamics is calculated by
Equation 4:
( )
F = Binomial Adf , P (C) (4)
where Adf is the total female mouse abundance and F is the
number of newly pregnant female mice resulting from one
day of mating. MouseGD still assumes panmixia, unless oth-
erwise specified, assigning each mating female to a mate
among the adult male population.
Then, Equation 5 calculates the number of newly gestating
mouse offspring resulting from one day of mating:
∑
F
G= Poisson(𝜆 = Q)i (5)
i
BAYESIAN NETWORK GENE DRIVE RISK ASSESSMENT
where D is the daily probability of surviving the density-
dependent process; K is the maximum population estimate
for the adult mice; Ad is the current adult mouse popula-
tion; and Θ represents a shape parameter used to limit the
mouse population to its maximum population estimate (K).
We used a Θ of 22.4 and a K of 50,000 for our simulations.
We assumed life stage times of 19 days for gestation, 23 days
for nursing, 37 days for adolescence, and 690 days for adult-
hood. We based M. musculus life cycle parameters on esti-
mates from Brust et al. (2015). At the time of publishing this
work, no density-dependent or density-independent mortality
is applied to the gestating, nursing, or adolescent M. musculus
life stages in MouseGD. However, abundances of each ado-
lescent life stage are calculated as part of daily mouse popu-
lation dynamics, therefore, such mortality processes could be
added to the model with relative ease.
In MosquitoGD, properties of gene drives are stored as 3-
dimensional arrays termed “Inheritance Cubes” which con-
tain the expected offspring frequencies for each genotype for
every possible mating pair, as well as, parameters to spec-
ify mechanisms such as the viability of specific mating pairs,
genotype-specific fitness reduction, and genotype-specific
sex emergence (Sánchez, Bennett et al., 2020; Sánchez, Wu
et al., 2020). In MouseGD, we added a novel inheritance
cube function for the Sox9 CRISPR-Cas9 gene drive (Figure
S2). We also included brodifacoum-induced mortality in the
daily population dynamics of MouseGD simulations, apply-
ing a daily probability of mortality to each adult mouse
using random draws from a binomial distribution following
Equation 7:
Adt = Binomial (Adt−1 , 1 − P (Daily Mortality)) (7)
where the probability of daily toxicity-induced mortality was
calculated from the brodifacoum dose–response functions
(Equations 1 and 2). We applied brodifacoum mortality for
56 consecutive days in simulations, varying the timing and
dose of brodifacoum application between management sce-
narios (Table S2).
Changes to the source code of MGDrivE version 1.5.0
(Sánchez, Bennett et al., 2020) were implemented using
RTools version 3.5.0.4 (Ripley et al., 2020), RStudio version
1.2.5024 (RStudio Team, 2020), and R version 3.6.3 (R Core
Team, 2020).
2.3.2 Initial conditions used for MGDrive
simulations
For each of the four gene drive release scenarios simulated for
this risk assessment, we assumed that the SEFI mouse pop-
ulation had a maximum population of 50,000 mice, an esti-
mate based on spatial M. musculus density data from the SEFI
EIS (USFWS, 2019a). We also assumed that the SEFI mouse
population was divided into two patches containing 80% and
20% of the total population, with a 20% daily migration rate
2841
from the small patch to the large patch and a 5% daily migra-
tion rate from the large patch to the small patch. We estimated
patch sizes based on a topographic elevation map of SEFI
from the EIS appendices (USFWS, 2019b). The four release
schemes for GDM mouse introduction (GD1, GD2, GD3,
GD4) were parameterized as described by Table S1. Addi-
tionally, we specified gene drive-specific parameters for the
Sox9 construct, defining the inheritance patterns for the pay-
load allele, wild-type allele, and resistance alleles. Descrip-
tions of alleles and gene drive parameters can be found in
Tables 1 and 2, respectively.
3 RESULTS
After simulating all management scenarios in MouseGD, we
compared changes is mouse abundance and eradication risk
in relation to gene drive homing rate, brodifacoum dose,
brodifacoum timing, and GD mouse release scheme. Then,
we assessed the relative influence of each variable on eradi-
cation risk using the results of our sensitivity analyses.
The minimum time-to-eradication for the Sox9 gene drive
was 5 years, with most eradications occurring between 7 and
10 years after the initial deployment of GDM mice. We evalu-
ated what variables drive the time-to-eradication and the over
all probability of eradication within a 10-year window using
our model results and BN sensitivity analysis. Simulation
results showed that the performance of the Sox9 gene drive
in eradicating the SEFI mouse population is highly depen-
dent on drive homing rate, suggesting that careful empirical
research into determining CRISPR gene drive homing and
resistance rates is essential to predicting the rate of successful
population suppression. The complete CPT for mouse abun-
dance is available in the Supporting Information (Table S3).
3.1 Risk to mouse eradication endpoint
We also examined how mouse eradication risk varied with
time, homing rate, and brodifacoum application across
all release schemes. The probability of mouse eradication
increased with homing rate across all release scenarios. No
simulations showed mouse eradication within 5 years for
95% homing rate, however 6-year eradication was shown
when supplemental rodenticide was used in conjunction with
a 95% homing rate gene drive. Regardless of homing rate
or pesticide use, all simulated scenarios showed an eradica-
tion probability of greater than 3.7% within 7 years of GDM
mouse release.
Mouse eradication probability also increased with pesti-
cide dose. Only scenarios with an assumed pesticide dose of
2.02 mg/kg showed any probability of eradication within 5
years of GDM mouse release. In all scenarios, deploying pes-
ticide at the start of GDM mouse deployment resulted in a
higher probability of eradication. For most scenarios across
most years, deploying pesticide at the end of GDM mouse
releases resulted in a lower eradication probability than if no
2842 BROWN ET AL.

TA B L E 1 Descriptions of each allele present in the simulated SEFI population, including the degree of fertility reduction resulting in organisms that are
homozygous or heterozygous for each allele. Fertility reduction from Sox9 gene drive (H) alleles is sex-specific

Homozygous % fertility Heterozygous % fertility

Allele Description reduction reduction

W Wild-type allele for M. musculus population on Southeast 0 0

Farallon Island (SEFI). This is the gene that can be deleted
and replaced by the Sox9 CRISPR-Cas9 homing gene drive.
H Gene drive allele resulting from successful homology-directed 0 (Males) 0 (Males) 100
repair (HDR) by the Sox9 CRISPR-Cas9 construct. This 100 (Females) (Females)
allele causes female (XX) mice to develop male
morphology, causing them to be sterile and to compete
against males (XY) for wild-type XX mates.
B Out-of-frame resistance allele. These alleles occur as a result of 100 90
frameshift mutations caused by non-homologous end joining
(NHEJ) or other random mutations to the target DNA
sequence, rendering the DNA sequence unrecognizable by
the homing gene drive.
R In-frame resistance allele. These alleles occur as a result of 0 0
frameshift mutations caused by NHEJ or other random
mutations to the target DNA sequence, rendering the DNA
sequence unrecognizable by the homing gene drive.

TA B L E 2 Descriptions of gene drive-specific parameters used in MGDrivE simulations. For the full mathematical context of these parameters, see the
MGDrivE equation at https://github.com/MarshallLab/MGDrivE

Values used in
Parameter Description simulations

Cutting rate (C)
Correct homing rate (CH)
Resistance generation rate (CR)
The probability of successful double-strand breakage (cutting) by the Cas-9 protein of 99.9%
the Sox9 CRISPR-Cas9 homing drive. If cutting is unsuccessful, the organism retains
its wild-type (W) allele.
The probability of successful homology-directed repair (HDR) given successful cutting 95%, 99%, 99.9%
of the wild-type allele. Results in organisms that are homozygous for gene drive (H)
alleles. If homing is unsuccessful, non-homologous end joining (NHEJ) occurs,
leading to resistance.
The probability that NHEJ results in an in-frame-resistant (R) allele. (1-CR) is the ̄
33.3%
probability of out-of-frame resistant (B) alleles.

rodenticide had been used. These findings suggest that if a
supplemental rodenticide is deployed with a gene drive, it is
best to deploy the rodenticide as early as possible in the eradi-
cation process, while wild mice still greatly outnumber GDM
mice.
dose had a higher influence on eradication. This suggests that
the level of pesticide used is an important determining fac-
tor in estimating the speed of a successful eradication, while
gene drive homing rate drives the overall probability of a suc-
cessful eradication.

3.2 Sensitivity analysis

In our sensitivity analyses (Table S3; Figure 3), we focused
on comparing the relative influence of gene drive hom-
ing rate, brodifacoum dose, and brodifacoum timing on the
probability of mouse eradication, comparing each variable’s
entropy reduction across release scheme and simulation year.
Overall, homing rate had a higher entropy reduction than the
addition of rodenticide meaning that, in the simulations we
ran, the resistance generation rate had more influence on the
risk of mouse eradication than the influence of toxicity. How-
ever, in years 5 and 6 after GDM mouse release, rodenticide
4 DISCUSSION
This BN-RRM risk assessment framework for gene drives
demonstrates that outcomes and uncertainties pertaining to
environmental applications of gene drives can be probabilis-
tically evaluated with clearly-documented modeling assump-
tions. We now relate this work with NASEM criteria for risk
assessment of gene drives, elaborating on how this model fits
into the broader context of synthetic biology and ecologi-
cal risk assessment, drawing conclusions, and outlining next
steps.
BAYESIAN NETWORK GENE DRIVE RISK ASSESSMENT
F I G U R E 3 Results of sensitivity analyses for GD1-4 at years 5 and 10 post-GDM mouse deployment. Percent entropy reduction represents the
influence of each variable on the risk of mouse eradication. We compared the influence of supplementary rodenticide, rodenticide timing, and gene drive
homing rate. Homing rate had higher entropy reduction in later years of simulations, while pesticide dose had higher influence in years 5 and 6. We used
Netica for all calculations
4.1 Alignment with NASEM criteria
Chapter 6 of Gene Drives on the Horizon (NASEM, 2016)
outlines some key properties that should be present in an eco-
logical risk assessment of gene drive technology:
1. The risk assessment should be able to provide prob-
abilistic estimates for potential harm and benefits of
gene drive deployment using numerical models.
BNs can combine quantitative modeling techniques and
empirical information into calculations of probability distri-
butions representing risk. The model demonstrates this prop-
erty by generating distributions of mouse abundance and
mouse eradication from gene drive simulations. BNs can
compare relative impacts to multiple endpoints due to the
influence of multiple stressors.
2. The model should allow for comparison between mul-
tiple management strategies.
The model contains the risk distributions from 60 simu-
lated gene drive management scenarios, showing their effi-
cacy across a 10-year period. The outcomes of these scenar-
ios were calculated to inform management goals by calculat-
ing risk to a site-specific endpoint. While the sole focus of
this study was quantifying the influence of gene drive param-
eters and rodenticide use on the risk of mouse eradication, as
demonstrated by previous BN-RRM publications (Eikenbary,
2020; and Landis, Chu et al., 2020), BNs can quantify risk to
multiple endpoints within the same model framework.
2843
3. The model should be able to incorporate the concerns
of relevant management, stakeholders, and publics.
The endpoint node in this BN reflects specific manage-
ment concerns the invasive mouse problem on SEFI, show-
ing the probability of accomplishing the management goal of
mouse eradication given various combinations of gene drive
and brodifacoum use. Landis et al. (2017) emphasizes the
ability of the BN-RRM to evaluate a wide range of endpoints.
These endpoints can be viewed in terms of their costs and
benefits to various stakeholder groups.
4. The model should have utility in identifying sources
of uncertainty and forming an adaptive management
scheme around gene drives.
We used sensitivity analysis to determine the relative influ-
ence of variables on the probability of mouse eradication on
SEFI. Within an adaptive management framework, sensitiv-
ity analyses can be used to prioritize research investigating
the efficacy of various gene drive constructs. The BN-RRM
can be continuously updated with new knowledge derived
from management decisions and new research (Landis et al.,
2017).
This work meets the recommended criteria for ecologi-
cal risk assessment of gene drives outlined by Gene Drives
on the Horizon chapter 6 (NASEM, 2016). The approach
highlights a framework for directing empirical gene drive
research and developing site-specific adaptive management
schemes for potential environmental applications of gene
drives.
2844
4.2 Risk assessment for synthetic biology
The properties of this risk assessment framework for gene
drives are broadly applicable to any ecological use of syn-
thetic biology. The usefulness of probabilistic estimates of
environmental outcomes is wide-ranging. Additionally, the
flexible and causal nature of the BN-RRM is suitable to a
plethora of potential scenarios related to emerging biotech-
nology. Using the MouseGD framework to parameterize this
BN allowed us to account for population dynamics, popula-
tion dynamics, patch dynamics, specific gene drive proper-
ties, and toxicology when simulating the SEFI mouse eradi-
cation. Adapting MouseGD to risk assessment demonstrated
the flexibility of the BN-RRM to incorporate novel numerical
modeling techniques into a causal BN framework.
4.3 Conclusions
We demonstrated an approach to parameterizing a risk assess-
ment model for the environmental deployment of GDM
organisms, providing a framework that can compare multiple
management strategies and isolate sources of scientific uncer-
tainty. The purpose of this framework is to assist in priori-
tizing research needs and developing adaptive management
strategies, enabling responsible investigations into the use-
fulness of gene drive technology and synthetic biology as a
whole.
In simulating the deployment of GDM mice on SEFI,
we found that the risk of successful mouse eradication was
greatly influenced by the resistance allele generation rate of
the simulated Sox9 gene drive. These results suggest that thor-
ough laboratory investigations into the homing properties of
gene drives are critical for predicting the likelihood of suc-
cessful population suppression using a CRISPR-based gene
drive. Simulations also showed that GDM mouse population
suppression takes place across potentially long time horizons
(7–10 years), meaning that such an approach may not be fea-
sible to management scenarios where a quick eradication is
necessary.
4.4 Next steps
The risk assessment model presented in this study serves as a
template for developing decision-making models and orient-
ing future research around synthetic biology and gene drives.
However, before risk assessment frameworks can be used
to direct management decisions in the field, they must be
tested and validated using supplemental experimentation and
monitoring. As we demonstrated with sensitivity analysis of
the BN, studies to evaluate the performance of gene drives
in terms of successful cutting and homing of the CRISPR-
Cas9 system would be useful in calculating the probabil-
ities of various outcomes in a field deployment of GDM
organisms.
BROWN ET AL.
Another set of variables that were not fully evaluated in the
BN were the conservation-based endpoints, or the impacts to
nontarget species on SEFI due to the deployment of a pesti-
cide or gene drive. For example, processes such as hybridiza-
tion or horizontal gene transfer could potentially lead to
the transfer of gene drives to nontarget species (NASEM,
2016). Pesticides can also cause nontarget toxicity via mul-
tiple routes of exposure, including predation of dosed organ-
isms and nontarget consumption of bait. Although such vari-
ables were not the focus of this work, other BN-RRM publi-
cations have incorporated species interactions and toxicity to
nontarget organisms (Landis et al., 2017; Landis, Chu et al.,
2020). Many other potentially confounding variables were
excluded from the analyses including seasonal and predatory
influence on mouse population dynamics; residual toxicity,
fate, and transport of brodifacoum; early life stage mortality
processes for mice; and potential differences in brodifacoum
sensitivity between mouse life stages and for newly-released
GDM mice. Connolly et al. (2021) describes 46 causal path-
ways outlining potential hazards of gene drive use, including
the possible interactions between gene drives, pesticides, and
other environmental factors. These pathways are important to
consider, especially in an IPM scenario, where multiple pest
eradication techniques are used.
The feasibility of a Sox9 gene drive mouse eradication is
also highly uncertain. As of October 2021, no peer-reviewed
publication has reported any empirical research using GDM
mice. Our MouseGD simulations assumed a phased release
of 50,000 transgenic mice onto SEFI, an amount equivalent
to the wild population. Until laboratory research is conducted
that focuses on the effective and resource-efficient synthesis
of GDM mice, the viability of such an undertaking, in terms
of both cost and labor, remains unknown.
Chapter 5 of Gene drives on the Horizon (NASEM, 2016)
outlines a phased testing approach for gene drives (Figure
S3). This process involves research and deployment phases
encapsulating preliminary lab and field testing, field deploy-
ment of GDM organisms, and continuous monitoring of post-
deployment impacts. This approach is intended to be iterative
and cautious, with criteria for revisiting research or deploy-
ment phases when new information or uncertainties arise.
Risk assessment is intended to inform this phased testing
approach at all stages, revealing research needs and guiding
adaptive management of gene drives. The BN-RRM is a well-
suited framework for informing the NASEM-outlined goal of
responsible and thorough methods for evaluating proposed
environmental uses of gene drives and synthetic biology.
AC K N OW L E D G M E N T S
We would like to acknowledge Jared Bennett and Héctor
Manuel Sánchez Castellanos (UC Berkeley – John Marshall
Lab) for their assistance with “MGDrivE” source code inter-
pretation and modification. We would also like to acknowl-
edge Andy Bunn (WWU – IES) for technical assistance
with package modification in R. Finally, we would like to
acknowledge Colter Lemons (WWU – IETC) for assistance
with GIS and April Markiewicz (WWU – IETC) for technical
BAYESIAN NETWORK GENE DRIVE RISK ASSESSMENT
assistance through the Institute of Environmental Toxicology
and Chemistry.
ORCID
Ethan A. Brown https://orcid.org/0000-0003-0827-4906
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