PNAS
2022 Vol. 119 No. 51 e2218020119
https://doi.org/10.1073/pnas.2218020119
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COMMENTARY
Gene drive-mediated population elimination for biodiversity
conservation. When you come to a fork in the road, take it
Bruce A. Hay
a
,1
and Ming Guo
b,c
Gene drive occurs when alleles of genes, multigene cassettes,
or large chromosomal regions are transmitted to fertile prog
-
eny at greater-than-Mendelian frequencies (50%). Gene drive
can be used to bring about population suppression or elimi
-
nation when the rate at which the drive element increases in
frequency outpaces a fitness cost induced by its presence,
and the population is driven to an unfit state. Much work has
focused on applications involving mosquito vectors of human
disease (1). Many other applications have their origin in the
global problem of invasive species (2), and thinking about how
to ameliorate the many harms they are associated with: food
insecurity, human disease, economic loss, environmental
degradation, and loss of biodiversity. Invasive species are a
major driver of species extinction (3), and island endemic
populations are particularly hard-hit. While islands constitute
only 6.7% of land area, they host 20% of species and 50% of
threatened species and account for 75% of known extinctions
since the European expansion (4). Mice and rats are a com
-
mon culprit (Fig. 1) The primary method for eliminating them
utilizes rodenticides. This approach can succeed (5), but the
economics and logistics do not scale well with island size.
Toxicants can also result in off-target effects on other species,
which often precludes their use on islands inhabited by
humans and livestock/companion animals. Gene drive–based
population suppression provides a solution that eliminates
toxicant-based harms and is more humane. It is also species
specific and in principle lower cost because it is self-sustaining
and takes advantage of the invaders’ tendency to seek out
conspecifics even in complex and remote environments.
Conversely, a gene drive element must also be unable to bring
about suppression in nontarget areas if some individuals
manage to “jump ship.” The use of gene drive for population
suppression thus involves a fundamental tension between
the goals of robust spread and confinement of the desired
outcome to the target area. Because islands are isolated, they
have been a major focus of research into contexts in which
gene drive for population suppression could be tested, to real
conservation benefit, while limiting the possibilities for effects
elsewhere. The organization GBIRd (
https://www.geneticbio
-
control.org
) provides an important forum for consideration
of these ideas. Work by Gierus, Birand, and colleagues
addresses both issues (6). It outlines a method by which island
populations of mice (but not other rodents) could be elimi
-
nated through gene drive. Importantly, the designs involved
contain features that ensure the drive element cannot bring
about population suppression in a nontarget population.
Population elimination using gene drive has been achieved
in laboratory cage populations of
Anopheles
mosquitoes (7)
using an approach adapted from the behavior of naturally
occurring selfish genetic elements known as homing
endonuclease genes (HEGs) (8). HEGs increase in frequency
by copying themselves from one of a pair of homologous
chromosomes to the other using homologous recombination,
Author affiliations:
a
Division of Biology and Biological Engineering, California Institute
of Technology, Pasadena, CA 91125;
b
Department of Neurology, UCLA David Geffen
School of Medicine, University of California, Los Angeles, CA 90095; and
c
Department of
Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine, University
of California, Los Angeles, CA 90095
Author contributions: B.A.H. and M.G. wrote the paper.
The authors declare no competing interest.
Copyright © 2022 the Author(s). Published by PNAS. This open access article is distributed
under
Creative Commons Attribution License 4.0 (CC BY)
.
See companion article, “Leveraging a natural murine meiotic drive to suppress invasive
populations,”
10.1073/pnas.2213308119
.
1
To whom correspondence may be addressed. Email: haybruce@caltech.edu.
Published December 13, 2022.
OPEN ACCESS
Fig. 1.
A Laysan albatross chick on Midway Atoll, prey for invasive mice.
(credit; Wesley Jolley, Island Conservation).
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following creation of a double-strand DNA break on the wild-
type homologue. The ability of a synthetic HEG to eliminate
a mosquito population depends on homing rates being very
high, ~95% or higher. When homing rates are more modest,
modeling predicts an internal equilibrium in which the rate
of spread is balanced by the fitness cost to carriers, leaving
a substantial remaining population (9). Unfortunately, germ
line homing rates measured in mice are (to date) too low to
be of use (10–12). Thus, gene drive–based approaches to pop
-
ulation suppression in mice must take a different path. Nature
has provided an interesting alternative in the form of a pow
-
erful, although quirky, naturally occurring drive element, the
t
haplotype.
The
t
haplotype is a male meiotic drive element (also
known as a segregation distorter; reviewed in ref. 13) studied
for almost 100 y. It spans over 40 Mb on chromosome 17
(1.5% of the mouse genome) but is inherited as a unit due
to the presence of multiple inversions, which suppress
recombination. When present in heterozygous males, sperm
that lack the
t
haplotype are disabled, resulting in transmis
-
sion of
t
to progeny males and females at rates that can
exceed 95% depending on the specific
t
allele. Transmission
through the female germ line is Mendelian. Multiple genes
have been identified as contributors to this behavior (13).
However, not enough is known for
t
-like drive to be reverse
engineered in mice or other species. In short, the
t
haplotype
is a found object, not well understood, but available for use
as a gene drive tool.
Early modeling suggested that
t
alleles like
t
w2
, which are
viable and fertile as homozygous females but sterile as
homozygous males, might be able to drive small populations
of mice to extinction through the creation of populations in
which all males are sterile
t
homozygotes. However,
t
w2
and
other
t
alleles are found in nature at modest frequencies,
indicating that things are not so simple. Recent work shows
that polyandry (mating of females with multiple males) in
wild mice is high, and
t
-bearing sperm in
t
/+ heterozygotes
(+ indicates wild-type chromosome 17) have decreased com
-
petitiveness in comparison with +/+ (14). These forces, and
homozygous male sterility, antagonize spread of alleles such
as
t
w2
to high frequency under many conditions (6).
How can the ability of
t
w2
to spread at super-Mendelian
frequencies be utilized even if it is unable to directly drive
the population to an unfit state? Gierus, Birand, and col
-
leagues proposed placing Cas9 and a gRNA at a neutral posi
-
tion within the
t
haplotype. In this hybrid gene drive element,
which they refer to as
t
CRISPR
, Cas9 and the gRNA cleave and
(hopefully) create loss-of-function (LOF) alleles in the male
germ line of the prolactin (
Prl
) gene, which is required for
female fertility. The goal with
t
CRISPR
is for
t
-based segregation
distortion in males to pump the Cas9/gRNAs cassette to high
frequency within the population. The latter, through cleavage
followed by inaccurate repair in males, will continuously pro
-
duce LOF alleles at the independently segregating
Prl
locus.
The hope is that the combination of
t
-based drive and
accumulation of
Prl
LOF alleles will drive the population to
an unfit state that contains a high frequency of infertile
homozygous
Prl
mutant females along with some frequency
of infertile homozygous
t
males. The combination of these
two effects, they propose, could eliminate populations under
a wider range of parameters than with
t
w2
alone.
Modeling of
t
CRISPR
in spatially explicit populations supports
this idea (6).
t
CRISPR
is also predicted to work well with levels
of cleavage and LOF allele creation (~80%) that are plausibly
achieved. As with other Cas9-based methods for population
suppression,
t
CRISPR
can fail if resistant alleles appear. These
are alleles, either naturally occurring or generated by inac
-
curate repair following cleavage, that are now uncleavable
(because the gRNA no longer base pairs com
-
pletely with the target) but still allow for the syn
-
thesis of a functional product. They are rapidly
selected for when in competition with low-fitness
LOF alleles. Their appearance can be prevented
or at least delayed by targeting the fertility gene
at multiple positions.
Modeling also identifies an important set of interactions
between
t
w2
and
t
CRISPR
when both are present in the same
population. Under these conditions,
t
CRISPR
is at a disadvantage
as compared with
t
w2
and is eventually lost from the popula
-
tion. This happens because
t
CRISPR
alleles—which are contin
-
uously generating
Prl
LOF alleles in the male germ line—find
themselves in dead-end homozygous LOF prolactin female
progeny more often than do
t
w2
alleles. An important impli
-
cation of this dynamic noted by the authors is that if Cas9 or
gRNAs mutate to inactivity, which will inevitably happen if
population extinction does not occur, the
t
CRISPR
allele will be
converted (functionally) to a
t
w2
allele. Once this happens (or
migration of
t
w2
-bearing individuals to the island occurs),
t
CRISPR
may lose its advantage. This will occur even if new versions
of Cas9/gRNAs targeting a different fertility gene are intro
-
duced into the
t
w2
haplotype—because they would still be
competing with existing
t
w2
. Multiplexing of Cas9 and the
gRNAs to bring about redundancy can forestall but not pre
-
vent this process. In short, with a
t
CRISPR
-based approach, it is
important to get population elimination right the first time as
there will not be a second chance once significant levels of
t
w2
are present in the population. This same modeling result also
has, however, the important positive corollary that movement
of some
t
CRISPR
individuals to a larger mainland will never result
in population suppression because
t
w2
alleles, which we know
exist in a harmonious balance with wild type in the wild, will
inevitably arise from
t
CRISPR
in large populations.
The authors test the
t
CRISPR
idea using a format in which a
gRNA targeting the
Prl
gene is inserted into the
t
w2
haplotype,
and Cas9, expressed under the control of a male germ line–
specific promoter, sits elsewhere. In a nutshell, when males
carry both constructs, segregation distortion is maintained
(95%), and indel creation (and thus hopefully LOF allele cre
-
ation) in
Prl
occurred in the male germ line at a respectable
frequency of 80%. Finally, analysis of previously published
pooled whole genome sequences from multiple islands (15)
shows that
t
alleles are often, although not always, absent.
These modeling and experimental results argue that
t
CRISPR
provides a path to elimination of invasive populations of mice
on some islands. The fact that
t
CRISPR
will inevitably break
down to
t
w2
also makes it biologically implausible that t
CRISPR
“Work by Gierus, Birand and colleagues outlines a
method by which island populations of mice
(but not other rodents) could be eliminated
through gene drive.”
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PNAS
2022 Vol. 119 No. 51 e2218020119
https://doi.org/10.1073/pnas.2218020119
3 of 3
could achieve a similar end on a mainland. Related to this
last point, the authors note that deliberate introduction of
t
w2
into a neighboring mainland of concern would (if it is not
already there) serve a similar blocking function. A conceptu
-
ally important additional method for limiting suppression to
a target island, that applies to all DNA sequence modifica
-
tion-based drives, focuses on targeting what are known as
locally fixed alleles—alleles that are fixed (present on both
alleles of all individuals in the population) on the target island
but not on the mainland (16). In the context of
t
CRISPR
, alleles
of a gene required for female fertility that are fixed on the
island but not on the mainland would be targeted. Mainland
alleles different from the fixed island alleles are (because
they are not recognized by the gRNAs used on the island)
functionally resistant alleles. Modeling shows that even a low
frequency of such alleles will prevent population suppression
(16). Sequence analysis has begun to identify target genes
for the locally fixed allele approach (15).
Where do we go from here? Multiple unknowns remain.
In particular, the life history of mice on target islands should
be explored to provide values for the many variables that
can influence drive outcome. Any
t
CRISPR
strain to be used will
also need to be backcrossed extensively into the target pop
-
ulation genetic background to maximize fitness. All this char
-
acterization is necessary so as not to end up in a situation in
which a
t
CRISPR
is released on an island, only to find some years
later that it is not quite good enough to bring about eradica
-
tion, leaving the island immune to further
t
-based suppres
-
sion strategies.
Finally, a few words about time. Gene drive is sometimes
portrayed in the popular press as a tool that will scythe its
way through a population like a hot knife through butter,
rapidly bringing about population elimination. This is not
the case. Modeling by Gierus, Birand, and colleagues esti
-
mates times to eradication of ~20 to 30+ y depending on
the values for the variables noted above (and of course the
t
CRISPR
introduction frequency). Thus, if a gene drive like
t
CRISPR
is introduced onto an island, there will be ample time
to monitor and learn from its dynamics. A real question is
if endangered species on some of these islands will be able
to hold on long enough to be the beneficiaries of this
technology.
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