Gene drive that results in addiction to a
temperature-sensitive version of an essential gene
triggers population collapse in
Drosophila
Georg Oberhofer
a
, Tobin Ivy
a
, and Bruce A. Hay
a,1
a
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125
Edited by Dana Carroll, The University of Utah, Salt Lake City, UT, and approved October 14, 2021 (received for review April 21, 2021)
One strategy for population suppression seeks to use gene drive
to spread genes that confer conditional lethality or sterility, pro-
viding a way of combining population modi
fi
cation with suppres-
sion. Stimuli of potential interest could be introduced by humans,
such as an otherwise benign virus or chemical, or occur naturally
on a seasonal basis, such as a change in temperature.
Cleave and
Rescue
(
ClvR
)sel
fi
sh genetic elements use Cas9 and guide RNAs
(gRNAs) to disrupt endogenous versions of an essential gene while
also including a
Rescue
version of the essential gene resistant to
disruption.
ClvR
spreads by creating loss-of-function alleles of the
essential gene that select against those lacking it, resulting in pop-
ulations in which the
Rescue
provides the only source of essential
gene function. As a consequence, if function of the
Rescue
, a kind
of Trojan horse now omnipresent in a population, is condition
dependent, so too will be the survival of that population. To test
this idea, we created a
ClvR
in
Drosophila
in which
Rescue
activity
of an essential gene,
dribble
, requires splicing of a temperature-
sensitive intein (TS-
ClvR
dbe
). This element spreads to transgene
fi
x-
ation at 23
°
C, but when populations now dependent on Ts-
ClvR
db
e
are shifted to 29
°
C, death and sterility result in a rapid population
crash. These results show that conditional population elimination
can be achieved. A similar logic, in which
Rescue
activity is condi-
tional, could also be used in homing-based drive and to bring
about suppression and/or killing of speci
fi
c individuals in response
to other stimuli.
gene drive
j
population suppression
j
sel
fi
sh genetic element
j
Drosophila
G
ene drive occurs when particular genetic elements are
transmitted to viable, fertile progeny at rates greater than
those of competing allelic variants or other parts of the genome
(reviewed in ref. 1). There has long been interest in the idea
that selfish genetic elements mediating gene drive could be
used to spread a fitness cost into a population, thereby bringing
about population suppression or elimination (2–5). Selfish ele-
ments known as homing endonuclease genes (HEGs), which
encode a site-specific nuclease (synthetic versions use RNA-
guided nucleases such as Cas9 to achieve site specificity), pro-
vide one approach to achieving this goal. A HEG is located at
the same site in the genome as its target site, thereby disrupting
it. When a HEG is present in heterozygotes, the wild-type
(WT) allele, which contains an intact target site, is subject to
cleavage. If this DNA break is repaired through homologous
recombination (HR) using the HEG-bearing chromosome as
the template, then the WT allele is converted to a HEG allele,
thereby bringing about an increase in the HEG copy number
(for a review, see ref. 1). The HEGs observed in nature use
multiple methods, including inteins and self-splicing introns,
that allow themselves to be copied into highly conserved
sequences in essential genes without disrupting the function of
these genes (6), thereby minimizing any associated fitness cost.
In contrast, in 2003 (3) Austin Burt recognizes that if an engi-
neered HEG inserts itself into a gene essential for viability or
female fertility, population suppression can result as the HEG
spreads and the frequency of loss-of-function (LOF) homozy-
gotes (bisex or female specific) increases (7–11). Other
approaches, some of which also utilize homing, seek to
decrease population fitness by driving it to an all-male state by
shredding the X chromosome during spermatogenesis (12–17).
Population suppression through homing can fail when homing
rates are low (7, 8) and/or repair of cleaved target sites in the
essential gene results in the creation of uncleavable but func-
tional (resistant) alleles (compare refs. 9, 10, and 18). Ecologi-
cal factors such as inbreeding and spatial structure may also
hinder suppression (19–24). These variables must be deter-
mined on a species- and locus-specific basis. Similar considera-
tions apply to the use of Y-linked X shredders, which must also
function when present on the highly heterochromatic Y
chromosome.
An alternative approach to species-specific population sup-
pression that utilizes gene drive but does not require homing or
sex ratio distortion works in two steps. The first utilizes gene
drive to spread through a population (population modification)
one or more transgenes (Cargo) that confer conditional lethal-
ity in response to the presence of an environmental stimulus
such as an otherwise benign chemical, infection with a virus,
prokaryote or fungus, diapause, or a change in temperature
(compare refs. 2, 4, 5, and 25). The second step, population
suppression, follows upon introduction of the environmental
stimulus. A central challenge with this approach, which has pre-
viously been envisioned as involving the activity of a dominantly
acting Cargo transgene (compare refs. 2, 4, 5, and 25) is how to
ensure the continued function of this (by definition) nonessen-
tial gene since LOF mutations that inactivate it will be strongly
selected for. Here, we sidestep this problem through a two-part
strategy. In the first,
Cleave and Rescue
–based gene drive
(
ClvR
) is used to bring about population modification. Impor-
tantly, a necessary consequence of
ClvR
drive is that survival or
fertility of all members of the modified population becomes
dependent on the activity of a
ClvR
-encoded version of an
This paper results from the NAS Colloquium of the National Academy of Sciences,
“
Life
2.0: The Promise and Challenge of a CRISPR Path to a Sustainable Planet,
”
held December
10
–
11, 2019, at the Arnold and Mabel Beckman Center of the National Academies of
Sciences and Engineering in Irvine, CA. NAS colloquia began in 1991 and have been
published in PNAS since 1995. The complete program and video recordings of presenta-
tions are available on the NAS website at
http://www.nasonline.org/CRISPR
. The collection of
colloquium papers in PNAS can be found at https://www.pnas.org/cc/the-promise-and-
challenge-of-a-crispr-path.
Author contributions: G.O. and B.A.H. designed research; G.O., T.I., and B.A.H.
performed research; G.O., T.I., and B.A.H. analyzed data; and G.O. and B.A.H. wrote
the paper.
Competing interest statement: The authors have
fi
led patent applications on ClvR and
related technologies (US Application No. 15/970,728 and No. 16/673,823; provisional
patent No. CIT-8511-P).
Published under the
PNAS license
.
1
To whom correspondence may be addressed. Email: haybruce@caltech.edu.
This article contains supporting information online at
http://www.pnas.org/lookup/
suppl/doi:10.1073/pnas.2107413118/-/DCSupplemental
.
Published November 29, 2021.
PNAS
2021 Vol. 118 No. 49 e2107413118
https://doi.org/10.1073/pnas.2107413118
j
1of9
GENETICS
COLLOQUIUM
PAPAER
Downloaded at California Institute of Technology on November 29, 2021
essential gene (the
Rescue
). The potential for conditional popu-
lation suppression is brought about by making the activity of
the
Rescue
conditional. Here, we achieve this by embedding
within the
Rescue
coding region an intein cassette that can only
be correctly spliced out of the encoded protein (thereby achiev-
ing
Rescue
activity) at a low, permissive temperature. This strat-
egy disfavors selection for escaper mutations that have lost the
conditional feature of
Rescue
activity since point mutations,
insertions, and deletions are most likely to result in loss of
intein function, thereby preventing the creation of a functional
Rescue
protein. Similar considerations apply for related strate-
gies in which the activity of the
Rescue
is made conditional
through incorporation of an N-terminal conditional degron
(
Discussion
). Regardless of the exact mechanism, the key points
associated with this LOF-based strategy for conditional sup-
pression are that survival of the population becomes dependent
on a drive element–encoded version of an essential gene (the
Rescue
), the
Rescue
is engineered to be conditional, and most
mutations (though not all;
Discussion
) in the sequences confer-
ring conditionality will lead to self-elimination because they
also disrupt
Rescue
function.
ClvR
Selfish Genetic Elements as a Tool for Conditional Population
Suppression.
ClvR
selfish genetic elements (26, 27) [also referred
to as toxin antidote recessive embryo (TARE) in a related
proof-of-principle implementation (28)] have two components
(Fig. 1
A
). The first is a DNA sequence–modifying enzyme such
as Cas9 and one or more guide RNAs (gRNAs). These consti-
tute the
Cleaver
, are expressed in the germline, and act in trans
to disrupt the endogenous version of an essential gene, creating
potentially lethal LOF alleles in the germline and in the zygote
due to maternal carryover of active Cas9/gRNA complexes.
The second is a recoded version of the essential gene resistant
to cleavage that acts in
cis
to guarantee the survival of those
who carry it (the
Rescue
). The lethal LOF phenotype manifests
itself in those who fail to inherit
ClvR
and have no other func-
tional copies of the essential gene, while those who inherit
ClvR
and its associated
Rescue
survive. In this way, as with many
other toxin-antidote–based selfish genetic elements found in
nature (reviewed in ref. 29) and created de novo (30),
ClvR
gains a relative transmission advantage that can drive it to
genotype (all individuals carry at least one
ClvR
allele at the
locus) or allele (all alleles at the locus carry
ClvR
) fixation by
causing the death of those who lack it (26–28, 31). Importantly,
once a
ClvR
element has spread to genotype fixation (and
unlike other selfish elements in nature), all endogenous WT
alleles of the essential gene have been eliminated through
cleavage and LOF allele creation. At this point, the only source
of essential gene function comes from
ClvR
itself—a form of
genetic addiction that creates a state of permanent genotype
fixation. In consequence, if function of the
Rescue
, a kind of
Trojan horse now omnipresent in a population, is condition
dependent, so too will be the survival of that population.
Seasonal temperature is an environmental variable that
could in principle be used to bring about conditional popula-
tion suppression in rapidly reproducing species in which there
are a number of generations each year. For more slowly repro-
ducing species, in which drive to genotype fixation (and
the co-occurring loss of endogenous essential gene function)
requires a longer interval, other mechanisms of bringing about
conditional lethality or sterility, presumably based on environ-
mental triggers introduced by humans throughout the target
population range, would be required (
Discussion
).
Drosophila
suzukii
, an invasive species of Europe, Asia, and North and
South America (32, 33), is one potential target for a
temperature-based approach to conditional suppression. It has
a number of generations per year and is often invasive in tem-
perate climates that experience large seasonal temperature
variations (34), providing opportunities for introducing a
temperature-dependent population bottleneck as a method of
suppression. As a proof-of-principle demonstration of this idea,
we sought to create a version of
ClvR
in
Drosophila mela-
nogaster
in which
Rescue
function is temperature sensitive (TS;
TS
-ClvR
). We show that a TS-
ClvR
element can successfully
spread a conditional
Rescue
into
Drosophila
populations at per-
missive temperatures. When populations now dependent on
this transgene are shifted to nonpermissive temperatures, they
rapidly become sterile and go extinct.
Results
Insertion of a TS-Intein into the
Drosophila
Essential Gene
dribble
(
dbe
) Results in Temperature-Sensitive LOF.
Traditional approaches
to generation of dominant or recessive TS mutations in essen-
tial genes in metazoans are laborious as they involve random
mutagenesis of whole genomes followed by large-scale screens
at different temperatures for otherwise fit TS mutants. As an
alternative, we sought to create TS versions of an essential
gene by introducing a TS version of an intein into the protein
coding sequences of
Rescue
transgenes within
ClvR
s previ-
ously shown to spread into WT populations (Fig. 1 and refs.
26 and 27). An intein is a protein-encoded autoprocessing
domain able to excise itself from a polypeptide and rejoin the
N- and C-terminal flanking sequences (exteins) to create a
WT version of the encoded protein (35). Importantly, once an
intein has been introduced into the coding sequence of an
essential gene and that version provides the only source of
essential gene function, loss of splicing activity through muta-
tion cannot be selected for since the nonspliced version is
nonfunctional.
The
Sce
VMA intein, which is located within the
Saccharo-
myces cerevisiae
vacuolar membrane ATPase, is able to excise
itself from a number of foreign proteins (36). TS versions of
Sce
VMA inteins have been isolated that allow spicing at a
range of low but not higher temperatures [ranging from 18 to
30
°
C (37, 38)]. A mechanistic requirement for successful intein
splicing is that the C-terminal extein starts with a cysteine resi-
due. Other less well characterized sequence contexts also regu-
late splicing efficiency (39–41). To determine if
ClvR Rescue
genes that contain the
Sce
VMA intein are functional, we gen-
erated six WT- and six TS-intein–bearing versions of
Rescue
transgenes for two previously described
ClvR
target genes that
each include three cysteines in their coding regions [
dribble
(
dbe
), in
ClvR
dbe
(27) and
technical knockout
(
tko
), in
ClvR
tko
(26);
SI Appendix
, Fig. S1
D
]. We tested the ability of intein-
bearing
Rescue
transgenes to provide essential gene function
by examining progeny of a cross between females heterozygous
for complete
ClvR
dbe
or
ClvR
tko
elements and males heterozy-
gous for the corresponding WT-intein
Rescue
(
Rescue
-INT
WT
)or
TS-intein
Rescue
(
Rescue-
INT
TS
) transgene (Fig. 2).
When present in females,
ClvR
dbe
and
ClvR
tko
cleave and cre-
ate LOF alleles of their target genes in the maternal germline
and the zygote with a frequency of
>
99.9% (26, 27). Thus, in
the absence of another source of
Rescue
activity, essentially all
viable progeny should be
ClvR
bearing (in an outcross, the 50%
that fail to inherit
ClvR
die because they lack a functional copy
of the essential gene). In contrast, if the
Rescue
-INT
WT
or
Res-
cue
-INT
TS
in heterozygous males is active,
∼
33% of viable
progeny should be non–
ClvR
-bearing (as compared with 0.1%
for a full element), and these should all carry the intein-bearing
Rescue
(Fig. 2). From crosses carried out at 23 and 27
°
C, we
identified one version of the
dbe Rescue
that retained function,
in which the intein was inserted N-terminal to cysteine 2 of the
dbe
coding sequence (
SI Appendix
, Tables S1 and S2
). The
dbe
Rescue
transgene carrying the WT-intein was functional at 23
and 27
°
C. The
Rescue
carrying the TS-intein was also
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j
PNAS
Oberhofer et al.
https://doi.org/10.1073/pnas.2107413118
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
Downloaded at California Institute of Technology on November 29, 2021
functional at 23
°
C but was largely (though not completely)
nonfunctional at 27
°
C (Fig. 3 and
SI Appendix
, Table S2
). Flies
carrying the
dbe Rescue-
INT
TS
construct were then used as a
genetic background in which to create flies carrying a
full
ClvR
dbe
-INT
TS
(referred to as TS-
ClvR
dbe
) drive element
carrying the other components found in
ClvR
dbe
(27). These
include Cas9 expressed under the control of the germline regu-
latory sequences from the
nanos
gene, four gRNAs targeting
the endogenous
dbe
locus expressed under the control of indi-
vidual U6 promoters, and an
OpIE
-
td-tomato
marker gene (
SI
Appendix
, Fig. S1
B
and
C
).
TS
-ClvR
dbe
Efficiently Creates LOF Alleles at Permissive Temperatures.
A TS-
ClvR
must be able to efficiently create LOF alleles at all
relevant environmental temperatures, and Cas9 activity has
been shown to be temperature sensitive, with reduced activity
at lower temperatures (42, 43). To test the ability of Cas9 to
create
dbe
LOF alleles at temperatures permissive for intein
splicing, we crossed heterozygous TS-
ClvR
dbe
females to
w
1118
(WT) males at 22
°
C and scored viable progeny for inheritance
of the TS
-ClvR
dbe
marker. If the TS-
ClvR
dbe
Cas9/gRNAs
successfully create
dbe
LOF alleles in the maternal germline
and in the early embryo, viable progeny should be largely or
exclusively TS-
ClvR
dbe
bearing.
ClvR
was present in 93.8% of
the offspring, a lower frequency than previously reported for
the original
ClvR
dbe
[
>
99% (27)], in which crosses were carried
out at 26
°
C. This is likely due to reduced Cas9 activity, since
similar tests with the original
ClvR
dbe
stock at 22
°
C also
resulted in a reduced drive inheritance of 95.9% (
SI Appendix
,
Table S3
).
To test whether escapers from the cross of female TS-
ClvR
dbe
/
+
to male
w
1118
had acquired functional resistance alleles (an
uncleavable but functional version of the endogenous
dbe
gene)
that could thwart drive, we carried out several tests (summarized
in
SI Appendix
,Fig.S2
). First, we backcrossed all 91 escaper
males (the 93 females were not tested because they were not vir-
gin) to heterozygous TS-
ClvR
dbe
females and found an average
drive inheritance rate in progeny of 86.1% (30 did not produce
offspring), indicating that the
dbe
locus was still sensitive to LOF
allele creation. After mating, DNA was extracted from all males
that were still alive and the
dbe
target locus sequenced. Results
of the sequencing analysis are summarized in
SI Appendix
,Data
S1. In short, all of the sequenced flies retained WT target sites at
the
dbe
locus. Mixed sequencing signals were observed at specific
sites from some individuals, probably indicating cleavage of only
one copy of the
dbe
locus. Second, as a way of getting a more
general population level feel for the possible existence of unde-
tected escapers or alleles at other loci that prevent LOF allele
creation—and thus depress drive and population suppres-
sion—we carried out 12 drive experiments that each began with
an individual escaper male of particular concern as the starting
material. In brief, we selected 12 of the above 61 fertile escaper
males that had a lower-than-average bias toward being TS-
ClvR
dbe
bearing. To provide a representative sample, we selected
at least one cross from each of the four original bottles (A to D).
For each of these 12 crosses (of a non–TS-
ClvR
dbe
-bearing
escaper male to heterozygous
+
/TS-
ClvR
dbe
–bearing females) we
collected all male and female offspring (nonvirgin, non-
ClvR
,and
ClvR
bearing), moved them to 12 bottles, and carried out multi-
generation drive experiments (see
SI Appendix
,Fig.S2
). All pop-
ulations attained TS-
ClvR
dbe
genotype fixation within three to
five generations. To determine if all WT
dbe
alleles had been ren-
dered LOF once genotype fixation of TS-
ClvR
dbe
was achieved, the
12 TS-
ClvR
dbe
populations were transferred again and incubated at
29
°
C. Each bottle (
∼
300 individuals) produced a few progeny, but
lethal
Cas9
3
3
Female gametes
male gametes
3
ClvR/+
TS-Rescue/+
Cas9
Rescue
3
Rescue
3
TS-Rescue
3
TS-Rescue
Cas9
Rescue
3
3
3
3
TS-Rescue
3
3
viable
viable
???
viable at permissive T
lethal at restrictive T
Fig. 2.
Crossing scheme to identify conditional
Rescue
transgenes. Mater-
nally inherited (red centromere) and paternally inherited (blue centro-
mere) third chromosomes are indicated.
A
Cas9/gRNA
Intein
conditional Rescue
B
C-Extein
N-Extein
Protein
splicing
COOH
H
2
N
functional
Rescue
Intein
Target
Target
mutations
Time
Collapse
Population size
Trigger
Fig. 1.
TS-
ClvR
design and concept. (
A
)TS
-ClvR
drive element comprised of Cas9/gRNAs targeting an essential gene and a recoded
Rescue
(not recog-
nized by Cas9/gRNAs), which includes a TS
-
intein within its coding region. After translation, the TS-intein can splice itself out to yield a functional
Rescue
protein. (
B
) Population suppression with a TS-
ClvR
.TS
-ClvR
–
bearing
fl
ies (red) are released into a WT population (yellow). The TS-
ClvR
sel
fi
sh element
spreads into the population at the cost of WT. Once the TS
-ClvR
element has reached genotype
fi
xation (all individuals in the population have at least
one copy of TS-
ClvR
), all functional endogenous copy of the essential gene targeted by TS
-ClvR
will have been mutated to LOF. At this point, the condi-
tional TS
-Rescue
within the
ClvR
element provides the only source of essential gene function in the population, making it subject to a collapse in response
to a temperature shift.
GENETICS
COLLOQUIUM
PAPAER
Oberhofer et al.
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
PNAS
j
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https://doi.org/10.1073/pnas.2107413118
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none of these produced progeny in the next generation, arguing
that all endogenous WT alleles of
dbe
had been eliminated.
All together, these results show that while
ClvR
drive
strength (as measured by LOF allele creation rate) is mod-
estly reduced at low temperature due to reduced levels of
Cas9/gRNA activity, this is not associated with the appear-
ance of resistant alleles that prevent drive (
SI Appendix
,
Data S1 and Fig. S2
for workflow).
TS-
ClvR
dbe
Flies Suffer a Temperature-Dependent Loss of Reproduc-
tive Output.
In order to bring about condition-dependent popu-
lation suppression following gene drive–based population
modification, carriers must experience a high fitness cost under
nonpermissive conditions. A major determinant of fitness is
reproductive output.
Dbe
is a gene whose product is required in
all proliferative cells (44). Thus, reproductive output is likely to
be a sensitive indicator of
dbe
function and the effects of dos-
age at different temperatures. To explore these topics, we char-
acterized the number of adult progeny produced by females
having two, one, or no copies of TS-
ClvR
dbe
. Because the only
viable progeny of
ClvR
-bearing mothers are also
ClvR
bearing
(with
ClvR
now providing the only zygotic source of essential
gene function), reproductive output of these females is a func-
tion of both maternal and zygotic TS-
ClvR
dbe
Rescue
activity at
different temperatures. We focused on females because popula-
tion numbers are generally thought to be limited by female fer-
tility, and adult sexual maturation and ongoing fertility requires
cell proliferation and growth of somatic and germline cells. In
contrast, young adult males already contain large numbers of
mature sperm, which also have a long functional lifetime once
deposited in the female reproductive tract (45). Consistent with
this, when TS-
ClvR
dbe
males, raised at 23
°
C, were transferred
to 29
°
C as young adults, they showed an age- and temperature-
dependent fertility loss similar to that of control w
1118
males
(
SI Appendix
, Tables S5 and S6
). For each cross involving
females, four replicate vials having five females and five males
(derived from flies raised at 22
°
C) were incubated at different
temperatures ranging from 23 to 29
°
C and transferred to fresh
vials every 2 d. The cumulative adult fly output from these
crosses over time is plotted in Fig. 3 (see also
SI Appendix
, Fig.
S3 for individual plots). At the low temperature of 23
°
C,
crosses between homozygous WT (
w
1118
) flies resulted in the
production of progeny at a roughly constant rate, with only a
modest drop off in production during days 10 to 12. The rate of
offspring production over time was similar for crosses involving
homozygous (non-TS)
ClvR
dbe
males and females and for
crosses between WT females and homozygous TS-
ClvR
dbe
males, in which progeny carry one WT version of
dbe
and one
TS version. In contrast, crosses between heterozygous TS-
ClvR
dbe
females and WT males produced fewer absolute num-
bers of progeny. This is expected since the
∼
50% of progeny
that fail to inherit TS-
ClvR
dbe
die due to lack of essential
gene function. The rate of adult offspring production also
decreased in the last two intervals (
SI Appendix
,Fig.S3
),
suggesting that in an otherwise LOF background, even at
permissive temperatures, the combination of one maternal
and zygotic copy of the
dbe Rescue
-INT
TS
results in some
age-dependent loss of fecundity.
At higher temperatures (25 to 27
°
C), the loss of reproduc-
tive output associated with maternal and zygotic dependence
on the TS-
ClvR
dbe
Rescue
was more dramatic. At 29
°
C, hetero-
zygous TS-
ClvR
dbe
females (the potentially viable progeny of
which are heterozygous for TS-
ClvR
dbe
) gave rise to no adult
progeny, while homozygous TS-
ClvR
dbe
females (the
potentially viable progeny of which are homozygous for TS-
ClvR
dbe
since their mothers were mated with homozygous
TS-
ClvR
db
males) produced only a few viable progeny. Prog-
eny production also ended somewhat prematurely at 29
°
C
for crosses in which the female parent was WT or
ClvR
dbe
bearing. However, this appears to be a general temperature
effect since the ability to produce progeny was lost at a simi-
lar rate for both sets of crosses. These results involving
crosses of
ClvR
dbe
/
+
females to
dbe Rescue
-INT
TS
males at
different temperatures, and data presented in
SI Appendix
,
Tables S3 and S4
show that individuals carrying TS-
ClvR
dbe
(the vast majority of which lack
dbe
function from the
♀TS-ClvR/TS-ClvR X ♂TS-ClvR/TS-ClvR
♀TS-ClvR/+ X ♂w
1118
♀ClvR/ClvR X ♂ClvR/ClvR
♀w
1118
X ♂TS-ClvR/TS-ClvR
♀w
1118
X ♂w
1118
A
CD
B
Fig. 3.
Cumulative adult
fl
y output at dif-
ferent temperatures: (
A
)23
°
C, (
B
)25
°
C,
(
C
)27
°
C, (
D
)20
°
C. Shown is the cumula-
tive adult progeny output of four replicates
in which
fi
ve females were crossed to
fi
ve
males over 12 d. Crosses were heterozygous
$
TS-
ClvR
dbe
/
þ
×
#
w
1118
in magenta, homo-
zygous
$
TS-
ClvR
dbe
/TS-
ClvR
dbe
×
#
TS-
ClvR
dbe
/TS-
ClvR
dbe
in yellow,
$
w
1118
×
#
TS-
ClvR
dbe
/TS-
ClvR
dbe
in violet,
$
w
1118
×
#
w
1118
(control) in blue, and the original non-TS
$
ClvR
dbe
×
#
ClvR
dbe
(control) in green.
4of9
j
PNAS
Oberhofer et al.
https://doi.org/10.1073/pnas.2107413118
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
Downloaded at California Institute of Technology on November 29, 2021
endogenous locus;
SI Appendix
,TableS3
) are reproductively
fit (as inferred by the number of TS-
ClvR
dbe
–bearing adult
progeny produced by TS-
ClvR
dbe
– bearing females) at lower
but not higher temperatures.
TS-
ClvR
dbe
Spreads to Transgene-Bearing Genotype Fixation at a
Permissive Temperature.
Population modification followed by
suppression requires that drive into a WT population succeed
at low, permissive temperatures. To test the ability of TS-
ClvR
dbe
to achieve this end, we carried out a gene drive experi-
ment at 22
°
C. To seed the drive, we crossed heterozygous
TS
-ClvR
dbe
males (
w
1118
; TS-
ClvR
dbe
/
+
)toWT(
w
1118
) females
to create a starting TS-
ClvR
dbe
allele frequency of 25% in four
replicate populations. Mated females were allowed to lay eggs
in a food bottle for 1 d and removed afterward. The drive
experiments were kept in a temperature-controlled incubator at
22
°
C. After
∼
16 d, most progeny had developed into adults,
which were then removed from the bottles, scored for the pres-
ence of the TS-
ClvR
dbe
marker (
td-tomato
), and transferred to
a fresh food bottle to repeat the cycle. Results of the drive
experiment are shown in Fig. 4
A
. The TS-
ClvR
dbe
construct
reached genotype fixation between 9 and 10 generations in all
four replicate drive populations, while a construct carrying only
the
dbe Rescue-
INT
TS
but no Cas9/gRNAs did not increase in
frequency. By generation 18, TS-
ClvR
dbe
allele frequencies
ranged from 93.2 to 97.6% (
SI Appendix
, Table S7
).
Populations in Which TS
-ClvR
dbe
Is Ubiquitous Undergo a Population
Collapse When Shifted to Elevated Temperature.
The goal of drive
with a TS-
ClvR
is ultimately to bring about a population crash
in response to an environmental temperature shift once LOF
allele creation associated with population modification has ren-
dered all members of the population dependent on the
Rescue
-
INT
TS
. As a test of this hypothesis, we followed the fate of
drive populations shifted to 29
°
C at generations 10, 12, 13, 16,
and 17. At each of these points, adults from the 22
°
C drive
population were allowed to lay eggs for 1 d at 22
°
C in order to
continue the drive and then moved to 29
°
C to allow egg laying
for a further 2 d. Adults were then removed and the fate of the
29
°
C populations followed, as with the drive populations kept
at 22
°
C(
SI Appendix
, Table S8
). Populations fixed for
ClvR
dbe
(control) individuals produce many adult progeny over six gen-
erations when continuously housed at 29
°
C (c.f.
SI Appendix
,
Table S9
). In contrast, populations of drive individuals—which
at this point are heterozygous or homozygous for TS-
ClvR
dbe
—-
give rise to only a few adult progeny per parent for one more
generation (c.f. gray line leading from the number of genera-
tion 10 individuals transferred to 29
°
C to the generation 11
adult progeny number). These latter adults were universally
sterile, resulting in population extinction in the next generation
(Fig. 4
B
).
Discussion
Our results show that gene drive can be used to spread a trait
conferring conditional lethality into an insect population, result-
ing in a population crash when the restrictive condition is experi-
enced, in this case a temperature shift. Additional Cargo genes,
designed to bring about some other phenotype such as preven-
tion of disease transmission prior to conditional population sup-
pression could also be included in such gene drive elements. The
implementation described herein used the
ClvR
gene drive
mechanism, which concurrently renders LOF endogenous copies
of an essential gene and replaces them with a TS version as
spread occurs. A similar outcome (drive followed by condition-
dependent suppression) could also be achieved using strategies
in which a HEG homes into an essential gene locus, thereby dis-
rupting its function, while also carrying a cleavage-resistant ver-
sion of the essential gene as a rescuing transgene (46–50), that
in this case is engineered to be temperature sensitive.
How do TS and related forms of conditional population sup-
pression compare with other transgene-based population sup-
pression approaches? Non-gene drive–based methods such as
sterile male release and its variants offer one strategy (51–53),
as does inundation with a dominant transgene that mediates
killing in response to an environmental trigger, either as a solo
entity or as a part of a multilocus self-limiting drive (54–57).
These methods are attractive because they are mechanistically
straightforward, but they will often have greater costs to imple-
mentation than drive-based approaches, since they are not self-
sustaining (see refs. 1 and 57 for a more detailed discussion of
these issues). As noted in the introduction, gene drive–based
approaches that utilize HEGs and X-shredding provide, alone
or in combination, a powerful way of bringing about population
suppression or elimination in mosquitoes (11, 15, 17). They
have a low threshold and can function in the presence of signifi-
cant fitness costs. However, both have molecular and/or cell
biological and genetic requirements that may only be met in
some species: high rates of homing for HEGs; X/Y males, and
the cell biological conditions that allow a transgene on the Y to
bring about cleavage of the X that results in a loss of X-bearing
sperm for X shredders. In addition, because drive in these sys-
tems results in the direct creation of genotypes that mediate
suppression—inviable or sterile homozygotes for HEGs; a male
bias for X shredders—other factors can in principle play roles
during drive to limit the suppression achieved for specific intro-
duction paradigms. These include population inbreeding, spa-
tial structure, and selection for mutations in trans (at other
loci) that suppress drive (19–24). The idea of modification fol-
lowed by conditional suppression, particularly with drive
AB
Fig. 4.
Population modi
fi
cation at a permissive temperature followed by suppression at a restrictive temperature. (
A
) Shown are genotype frequencies
of TS-
ClvR
dbe
–
bearing
fl
ies over discrete generations at 22
°
C. TS-
ClvR
dbe
is indicated with solid lines (replicates in different colors),
dbe Rescue-
INT
TS
con-
trols with dashed lines. (
B
) Gray lines show individual population trajectories for all replicates when incubated at 29
°
C. All populations produced some
offspring when moved from 22 to 29
°
C. These collapsed in the next generation due to complete sterility.
GENETICS
COLLOQUIUM
PAPAER
Oberhofer et al.
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
PNAS
j
5of9
https://doi.org/10.1073/pnas.2107413118
Downloaded at California Institute of Technology on November 29, 2021
elements such as
ClvR
that do not utilize homing to bring about
their spread, provides an alternative paradigm, in which drive
mediating population modification is separated in time from
suppression. In consequence, in such systems (and in HEG-
based systems that carry a TS-Rescue version of the essential
gene disrupted by the HEG), there is likely to be minimal selec-
tion, during drive, for mutations in cis or trans that block the
population suppression mechanism. This positive feature of
conditional suppression notwithstanding, non-HEG and
X-shredding drive mechanisms such as
ClvR
and various forms
of underdominance will typically come with some kind of intro-
duction threshold and spread more slowly than HEGs (in
organisms with a high frequency of homing), particularly when
rare (1). In addition, effective conditional suppression is contin-
gent on being able to introduce the trigger at the right time and
place. Thus, conditional suppression based on temperature sen-
sitivity (TS-
ClvR
or engineered HEG with a TS-
Rescue
)
requires a temperate climate and is only possible in species
(often insects) that have many generations per year, sufficient
to bring about drive to genotype fixation prior to onset of a
restrictive temperature. Similar points apply to other seasonal
variables such as humidity and daylight length. For organisms
with a slower reproductive rate, conditional suppression follow-
ing drive will require different stimuli, probably introduced
throughout the target area by humans (though see later in this
section for a discussion of strategies that utilize conditional kill-
ing following natural infection to eliminate only individuals that
carry a specific human pathogen). The challenge with human-
introduced triggers for population suppression is that they must
be dispersed in such a way as to make timely contact with all
individuals in the target population. In the case of a chemical
trigger, this condition could apply in already managed environ-
ments (agriculture) or perhaps enclosed bodies of water.
Viruses, bacteria, and fungi (see discussion below for an exam-
ple of how a virus might act as a trigger) provide other poten-
tially more self-sustaining and disseminating triggers for larger
areas.
The conditional population suppression system described
herein targets both males and females since
dbe
gene activity is
required in both sexes. In such a system, complete suppression
(population elimination) in response to a temperature shift is
possible when
ClvR
-dependent drive results in transgene-
bearing genotype fixation. This is because at this point, all
endogenous alleles of the essential gene have been eliminated
from the population and essential gene function derives only
from the conditional
Rescue
. With such a system, the target
environment may require some level of periodic repopulation
with transgenic individuals as a result of incoming migration of
WT. A modified system that would reduce this need, and work
to maintain the transgene in the target environment in the face
of incoming migration of WT, eliminates only females or female
fertility under nonpermissive conditions (for modeling of a
related system with these characteristics see ref. 58).
ClvR
s that
bring about LOF and
Rescue
of two different genes, one that is
needed for unconditional and sex-independent viability (medi-
ating strong drive) and a second that is conditional and
required for female viability or fertility (allowing for elimina-
tion of only females under nonpermissive conditions), could be
used to achieve this goal. Drives that carry a version of the sex
determination gene
doublesex
(
dsx
) with a TS-intein in the
female-specific exon provide one possible implementation.
ClvR
s able to rescue the viability and fertility associated with
LOF of two different essential genes at the same time have
been created (26, 27), suggesting this approach is plausible.
Finally, we note that the strategy for generating TS strains
described here (replacement of a WT version of an endogenous
gene with a TS version) could also be used as a method of engi-
neering sex-specific sorting in inundative suppression strategies
such as the sterile insect technique. Many existing SIT systems
utilize strains in which TS sex sorting can be carried out (59).
That said, these successes were hard won. TS strain creation
involved large-scale mutagenesis and screening of many thou-
sands of genotypes, followed by introgression of the TS trait
into often complex and specific genetic backgrounds that must
be otherwise fit. In many cases, the mutations that result in TS
sensitivity remain unknown, limiting the ability to move the
trait into related species (59). In cases in which the nature of
the TS mutation is known (as for a number of TS mutations in
D. melanogaster
), the ability to create similar (hopefully also TS
lethal) mutations in pest species using Cas9 provides one way
to simplify this process (59), though the fitness costs associated
with these changes can only be discovered empirically (60).
Success with any TS gene drive system in the wild will
require knowledge of temperature fluctuations within a season
in the region of interest, the life phases in which the target spe-
cies is most susceptible (and resistant) to loss of essential gene
function, and potentially further selections in rapidly reproduc-
ing organisms like yeast (37, 38) for TS-inteins best suited to
the environmental temperature regimes involved. Also, because
seasonal temperatures do not change in an all or none fashion,
gradual shifts toward nonpermissive conditions will provide
opportunities for selection to take place on sequences within
the intein coding region that reduce or eliminate temperature
sensitivity. The targeting of biosynthetic essential genes such as
dbe
, whose transient LOF is unlikely to result in an immediate
fitness cost (as is seen for some other TS mutants that cause
immediate paralysis; c.f. ref. 61) probably provides some level
of environmental phenotypic buffering in this regard but would
not eliminate selection. While next-generation
ClvR
elements
can be cycled through a population, replacing old, failed ele-
ments with new ones (27), strategies that forestall the need for
such cycles of modification for as long as possible would be use-
ful. This can be achieved by building into the
Rescue
transgene
mechanistic redundancy with respect to how temperature sensi-
tivity is achieved, thereby necessitating multiple mutational hits
for the
Rescue
to lose its TS characteristic. As an example, an
N-terminal TS degron (the N-terminal location preventing the
loss
of degron activity through frameshift or stop codons) that
promotes the degradation of a linked C-terminal protein at ele-
vated temperature provides one such approach (62). Insertion
of multiple copies of a common TS-intein at different positions
provides another.
Regardless of the mechanisms by which redundancy in terms
of conditionality are built into a drive system, it will be impor-
tant to determine the frequency with which mutations in cis
(within the TS-
Rescue
) and in trans (in other genes) arise that
can lead to
Rescue
function that is no longer conditional (sup-
pressor mutations) and if such mutations are present in wild
populations. Similar considerations also apply for non-TS con-
ditional systems. In the case of TS-
ClvR,
dominant suppressor
mutations can be identified by mutagenizing males of the TS
strain and crossing these to females also of the TS strain (in
which all endogenous copies of the essential gene have been
rendered LOF) at the permissive temperature. If progeny
raised at the nonpermissive temperature survive and are fertile,
a suppressor mutation has likely been created. Recessive cis-
and transacting suppressors can be identified in similar, albeit
more labor-intensive screens. Screens in yeast similar to those
used to identify the original TS-intein (37, 38) can also be uti-
lized to identify cis-acting suppressor (revertant) mutations. In
addition, it will also be important to explore the behavior of TS
ClvR
s in populations that originate from diverse ecologies, par-
ticularly with respect to environmental temperature range,
since these populations could have floating within them alleles
of genes encoding proteins such as heat shock proteins/chaper-
ones that may—as a byproduct of activities needed to promote
6of9
j
PNAS
Oberhofer et al.
https://doi.org/10.1073/pnas.2107413118
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
Downloaded at California Institute of Technology on November 29, 2021
normal protein folding at elevated temperatures—also function
as suppressors of TS lethality by promoting the folding and/or
stability of TS proteins.
Finally, we note that a similar logic to that presented here, in
which
Rescue
activity is conditionally blocked, could be used to
bring about species-specific suppression in response to other
stimuli. Small molecules provide one example. These could
block intein splicing activity (63), promote the degradation of a
target protein (64), or decrease the stability of specific tran-
scripts (65). Target genes that might be particularly amenable to
such approaches, which will likely alter expression only transiently
following application, include those encoding proteins whose loss
results in rapid cell death, such as inhibitors of apoptosis (66).
Virus infection provides a further opportunity for engineering
conditional lethality. As an example, virus-encoded protease activ-
ity, required for viral polyprotein processing in many systems,
serves as an “honest” and specific indicator of infection. If one or
more viral protease target sites are engineered into the products
of key host essential genes—and these versions replace WTcoun-
terparts during drive—cleavage at these sites in organisms that
are virally infected could result in a lethal LOF phenotype. This
could be used to directly suppress populations in response to
introduction of a naturally occurring and otherwise benign virus.
A similar strategy could also be used to selectively eliminate mem-
bers of a disease vector population that are infected with a human,
animal, or plant pathogenic virus in the context of a simple popu-
lation modification scenario.
Materials and Methods
Synthesis of TS
-Rescues
for
tko
and
dbe
Target Genes.
All constructs in this
work were assembled with Gibson cloning (67). Enzymes were from NEB and
cloning and DNA extraction kits from Zymo. Inteins were gene synthesized as
gblocks from IDT. We started from our previously cloned
Rescue
constructs
(26, 27). The
Rescue
for
tko
was derived from the ortholog of
Drosophila virilis
,
the one for
dbe
from
D. suzukii
. Both genes have three cysteines in their cod-
ingsequences.WeusedGibsonassemblytoinsertaWT-inteinandaTS-intein
[mutation D324G (37, 38)] after each of the cysteines for a total of 12 con-
structs. In addition, the plasmids had a dominant
OpIE
–
green
fl
uorescent
protein (GFP) marker, an attP site, and homology arms to facilitate CRISPR-
HR
–
mediated insertion into the
fl
y genome at the 68E map position on chro-
mosome 3.
The constructs were injected into
w
1118
fl
ies along with a preloaded Cas9/
gRNA RNP complex having a gRNA (both from IDT) targeting chromosome 3 at
68E (
SI Appendix
,Fig.S1
A
). Details were as described previously (27). All Gibson
cloning primers and construct GenBank
fi
les are in
SI Appendix
,DataS1
.
Embryonic injections were carried out by Rainbow Transgenic Flies. Injected G0
fl
ies were outcrossed to
w
1118
and screened for ubiquitous GFP expression.
Screening Crosses for Temperature-Dependent
Rescue
Activity.
To determine
if any of the intein-bearing
Rescues
showed temperature-dependent
Rescue
activity, we set up crosses between heterozygous virgins that carry the original
non-TS
ClvR
element and heterozygous males carrying the different
Rescue
-
INT
(TS or WT)
versions (crossing scheme in Fig. 2). All crosses were set up in tripli-
cates and incubated at 23 or at 27
°
C. None of the intein-
Rescues
for
tko
were
able to provide adequate gene function at either temperature (
SI Appendix
,
Table S1
). For
dbe
,the
Rescue
transgenes carrying the
WT
-intein inserted after
cysteine 2 and 3 were able to rescue
fl
ies at both temperatures.
Rescue
trans-
genes containing the TS-intein inserted after cysteines 1 or 3 were not able to
provide
Rescue
function at either temperature. In contrast,
Rescue
transgenes
carrying the TS-intein inserted after cysteine 2 showed promising behavior,
with most progeny dying at 27
°
C but not at 23
°
C(
SI Appendix
,TableS2
,
highlighted in red). We used these
fl
ies to build a fully functional TS-
ClvR
self-
ish element. Note: For the WT-intein inserted after cysteine 1 of
dbe
,wedid
not obtain transformants after a
fi
rst round of injections. Since the TS-intein
version of that construct did not show
Rescue
activity, this insertion position
was not further pursued.
Synthesis of
TS-ClvR
dbe
Flies.
Cas9 and a set of four gRNAs (each driven by a
U6 promoter) that target endogenous alleles of
dbe
were integrated into the
attP site within the TS-intein
Rescue
construct, as described previously (26, 27).
The gRNA scaffolds were optimized as described previously by replacing the T
base at position 4 with a G and extending the duplex by 5 bp (68, 69).
The construct was modi
fi
ed further using Gibson assembly to add in a new
OpIE-td-tomato
marker gene (the original plasmid had a
3xP3
-GFP marker
that would have been hard to screen for in the ubiquitous GFP background of
the TS
-Rescue
–
carrying
fl
ies) and was injected into
fl
ies carrying the TS
-Rescue
alongside a helper plasmid providing a source of PhiC31 integrase (Rainbow
Transgenic Flies) (
SI Appendix
, Fig. S1
B
). Injected G0
fl
ieswereoutcrossedto
w
1118
and screened for ubiquitous
td-tomato
expression. Positive transform-
ants were balanced over TM3,
Sb
to subsequently generate a homozygous
stock of TS
-ClvR
dbe
fl
ies carrying the TS
-Rescue
and Cas9/gRNAs (
SI Appendix
,
Fig. S1
C
). Primers and construct GenBank
fi
les are in
SI Appendix
,DataS1
.
Crosses to Determine Cleavage to LOF of TS
-ClvR
dbe
.
We crossed homozygous
TS
-ClvR
dbe
and
ClvR
dbe
(control) males to
w
1118
virgins to generate heterozy-
gous offspring. Heterozygous TS
-ClvR
dbe
(or
ClvR
dbe
control) virgins were
crossed to
w
1118
males, incubated at a permissive temperature of 22
°
C, and
the offspring was scored for the presence of the dominant TS
-ClvR
dbe
marker.
Results are shown in
SI Appendix
,TableS3
.
Analysis of Escapers.
From the experiment to determine cleavage to LOF, we
recovered 91 males that did not carry the TS
-ClvR
dbe
marker. We also recov-
ered 72 males that did not carry the
ClvR
dbe
marker from the control crosses
with the original
ClvR
dbe
fl
ies. All of them were crossed to heterozygous TS
-
ClvR
dbe
/
+
(or
ClvR
dbe
for the controls) females and incubated at 22
°
C again.
After they mated, we took the male out of each vial and extracted genomic
DNA. We ampli
fi
ed an amplicon spanning all four cut sites within the endoge-
nous
dbe
locus and sequenced it. The offspring of the crosses was again scored
for the presence of the TS
-ClvR
dbe
(or
ClvR
dbe
) marker. Afterward, we selected
12 vials with low cleavage to LOF rates and transferred all the offspring to a
food bottle to start a gene drive experiment as described below. In these gene
drive experiments, we did not score marker frequencies. The drive experiment
was continued until TS
-ClvR
dbe
(or
ClvR
dbe
controls) reached genotype
fi
xation
in all bottles. This took from three to
fi
ve generations. Bottles with TS
-ClvR
dbe
were subsequently transferred again and incubated at 29
°
Ctotestifapopu-
lation collapse could be induced. All results with a more detailed description
are shown in
SI Appendix
,DataS1
. The populations did crash, indicating that
no functional endogenous alleles exist in these drive populations.
Crosses to Test for Temperature-Dependent
Rescue
Function of TS
-ClvR
dbe
.
We set up crosses involving females and males (all reared at 22
°
C) of the fol-
lowing genotypes: homozygous TS
-ClvR
dbe
(10 vials),
w
1118
(control, 5 vials),
and
ClvR
dbe
(control, 5 vials). These were incubated at a potentially restrictive
temperature of 29
°
C. Offspring output of generations F1 and F2 are shown in
SI Appendix
,TableS4
.
Crosses to Determine Fecundity of TS
-ClvR
dbe
Flies over a Range of
Temperatures.
We set up
fi
ve different crosses (genotypes below). These
included
fi
ve females and
fi
ve males (four replicates) that had been reared at
22
°
C. After setting up the cross, the vials were incubated at 23, 25, 27, and
29
°
C. Every 48 h, adults were transferred to a fresh food vial, and this was
repeated
fi
ve times. We scored the adult
fl
y output in each of these vials.
Results are shown in Fig. 3 and
SI Appendix
,Fig.S3
. Crosses were as follows:
$
TS
-ClvR
dbe
/
þ
×
#
w
1118
,
$
TS
-ClvR
dbe
/
TS
-ClvR
dbe
×
#
TS
-ClvR
dbe
/
TS
-ClvR
dbe
,
$
w
1118
×
#
TS
-ClvR
dbe
/
TS
-ClvR
dbe
,
$
ClvR
dbe
×
#
ClvR
dbe
(control), and
$
w
1118
×
#
w
1118
(control).
Gene Drive Experiment.
We seeded four replicate populations by crossing het-
erozygous TS-
ClvR
dbe
/
+
males (or
Rescue
-INT
TS
/
+
that do not have Cas9/gRNAs
as a control) to
w
1118
females (25% starting allele frequency). Flies were
placed in food bottles, incubated at 22
°
C, and allowed to lay eggs for 1 d.
Afterward, they were removed from the bottles, and the eggs were allowed
to develop into adults. After
∼
16 to 17 d, a large number had eclosed
as adults. These were anesthetized on a CO
2
-pad, scored for the dominant
TS-
ClvR
dbe
marker, and transferred to a fresh food bottle to repeat the cycle.
Counts are in
SI Appendix
,DataS1
.
TS-
ClvR
dbe
and
ClvR
dbe
(Control) Populations at 29
°
C.
After the TS
-ClvR
dbe
fl
ies in the gene drive experiment reached genotype
fi
xation (generation 10
and following), we
fi
rst transferred them to a fresh food bottle to continue
the gene drive experiment. After they laid eggs in that bottle for 1 d, we
transferred them again to a fresh bottle. That second bottle was now incu-
bated at 29
°
C. Flies were given 2 d to lay eggs in that bottle before they were
removed again. Eggs were allowed to develop into adults that were then
scored and put in a fresh food bottle that was again kept at 29
°
C. Flies were
GENETICS
COLLOQUIUM
PAPAER
Oberhofer et al.
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
PNAS
j
7of9
https://doi.org/10.1073/pnas.2107413118
Downloaded at California Institute of Technology on November 29, 2021
kept in that bottle for 1 wk prior to removal, so as to maximize the number of
eggs laid. However, no progeny developed within these bottles. Results are
shown in Fig. 4
B
(gray lines) and
SI Appendix
,TableS8
.
As a control experiment, we used the previously characterized
ClvR
dbe
stock, which carries a WT copy of the recoded Rescue (27).
ClvR
dbe
fl
ies were
taken from a gene drive experiment [generation 44 (27)], transferred to a
fresh food bottle, and incubated alongside the TS
-ClvR
dbe
bottles at 29
°
C.
They were allowed to lay eggs for 2 d, after which adults were removed. After
the eggs developed into adults, we determined the adult population number
and transferred these individuals to a fresh food bottle to repeat the cycle.
This was repeated for a total of six transfers with no obvious reduction in pop-
ulation size. Results are shown in
SI Appendix
,TableS9
.
Data Availability.
A ll study data are included in the article and/or supporting
information.
ACKNOWLEDGMENTS.
Stocks obtained from the Bloomington Drosophila
Stock Center (NIH P40OD018537) were used in this study. This work was
carried out with support to B.A.H. from the US Department of Agricul-
ture, National Institute of Food and Agriculture (NIFA) specialty crop
initiative under US Department of Agriculture NIFA Award No. 2012-
51181-20086 and the California Institute of Technology (Caltech) Resnick
Sustainability Institute. G.O. was supported by a Baxter Foundation
Endowed Senior Postdoctoral Fellowship and the Caltech Resnick
Sustainability Institute. T.I. was supported by NIH Training Grant No.
5T32GM007616-39.
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GENETICS
COLLOQUIUM
PAPAER
Oberhofer et al.
Gene drive that results in addiction to a temperature-sensitive version of an essential
gene triggers population collapse in
Drosophila
PNAS
j
9of9
https://doi.org/10.1073/pnas.2107413118
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