1
Title:
2
Gene drive that results in addiction to a temperature sensitive version of an essential gene
3
triggers population collapse in Drosophila
4
5
Authors:
6
Georg Oberhofer
1
* (ORCID:0000-0003-0930-1996), Tobin Ivy
1
(ORCID:
7
0000-0002-9116-3854) and Bruce A. Hay (ORCID: 0000-0002-5486-0482)
1
8
Affiliations:
9
1
California Institute of Technology. Division of Biology and Biological Engineering.
10
1200 East California Boulevard, MC156-29, Pasadena, CA 91125
11
*corresponding author;
oberhofer.georg@outlook.com
12
0
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
13
ABSTRACT:
14
One strategy for population suppression seeks to use gene drive to spread genes that confer
15
conditional lethality or sterility, providing a way of combining population modification with
16
suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise
17
benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature.
18
Cleave and Rescue
(
ClvR
) selfish genetic elements use Cas9 and gRNAs to disrupt endogenous
19
versions of an essential gene, while also including a
Rescue
version of the essential gene resistant
20
to disruption.
ClvR
spreads by creating loss-of-function alleles of the essential gene that select
21
against those lacking it, resulting in populations in which the
Rescue
provides the only source of
22
essential gene function. In consequence, if function of the
Rescue
, a kind of Trojan horse now
23
omnipresent in a population, is condition-dependent, so too will be the survival of that
24
population. To test this idea we created a
ClvR
in
Drosophila
in which
Rescue
activity of an
25
essential gene,
dribble
, requires splicing of a temperature-sensitive intein (TS-
ClvR
dbe
). This
26
element spreads to transgene fixation at 23
O
C, but when populations now dependent on
27
Ts-
ClvR
db
e
are shifted to 29
O
C death and sterility result in a rapid population crash. These results
28
show that conditional population elimination can be achieved. A similar logic, in which
Rescue
29
activity is conditional, could also be used in HEG-based drive, and to bring about suppression
30
and/or killing of specific individuals in response to other stimuli..
31
KEY WORDS
32
Gene drive, Drosophila, selfish genetic element, population suppression
33
1
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
34
SIGNIFICANCE STATEMENT
35
Gene drive can be used to spread traits of interest through wild populations. In some contexts the
36
goal is to suppress or eliminate the population. In principle, one way to achieve this goal is if the
37
trait being spread confers on carriers conditional lethality in response to an environmental
38
stimulus that is either introduced by humans into the target area at a specific time (a virus,
39
otherwise benign chemical; a kind of species-specific insecticide), or that occurs naturally on a
40
seasonal basis, such as a change in temperature. Here we show that
ClvR
selfish elements can be
41
used to spread a gene that confers lethality and sterility in response to increased temperature,
42
demonstrating that conditional population elimination can be achieved.
43
2
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
44
Introduction
45
Gene drive occurs when particular genetic elements are transmitted to viable, fertile progeny at
46
rates greater than those of competing allelic variants or other parts of the genome (reviewed in
47
(1) ). There has long been interest in the idea that selfish genetic elements mediating gene drive
48
could be used to spread an unconditional or conditional fitness cost into a population, thereby
49
bringing about population suppression or elimination (2–5) . Selfish elements known as homing
50
endonuclease genes (HEGs), which encode a site-specific nuclease (synthetic versions use
51
RNA-guided nucleases such as Cas9 to achieve site-specificity), provide one approach to
52
achieving this goal by spreading an unconditional fitness cost (6–10) . Other approaches, some of
53
which also utilize homing, seek to drive the population to an all-male state by shredding the X
54
chromosome during spermatogenesis (11–15) . Population suppression through homing can fail
55
when homing rates are low (6, 7) , and/or repair of cleaved target sites in the essential gene results
56
in the creation of resistant alleles (c.f. (8, 9, 16) ), variables that must be determined on a species-
57
and locus-specific basis. Similar considerations apply to the use of Y-linked X shredders, which
58
must also function when present on the highly heterochromatic Y chromosome.
59
60
An alternative approach to species-specific population suppression that does not require homing
61
or sex ratio distortion utilizes gene drive to spread through a population (population
62
modification) one or more transgenes that confer conditional lethality in response to a change in
63
an environmental variable such as the presence of an otherwise benign chemical, infection with a
64
virus, prokaryote or fungus, diapause or a change in temperature (c.f. (2, 4, 5, 17) ). A central
3
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
65
challenge with this approach is how to ensure the continued function of the (by definition)
66
non-essential Cargo gene or genes needed to bring about conditional lethality or sterility, since
67
loss-of-function (LOF) mutations that inactivate these components will be strongly selected for.
68
An approach that eliminates the possibility of transgene inactivating mutations resulting in loss
69
of condition-dependent lethality, and that we implement here, uses gene drive to make the
70
survival of individuals under permissive conditions – as a necessary consequence of gene
71
drive-based population modification – dependent on the activity of an essential gene engineered
72
to lack function under non-permissive conditions.
73
74
Cleave and Rescue
(
ClvR
) selfish genetic elements as a tool for temperature sensitive
75
population suppression.
To achieve these ends, we sought to develop condition-dependent
76
versions of the
Cleave and Rescue
(
ClvR
) selfish genetic element (18, 19) (also referred to as
77
toxin antidote recessive embryo (TARE) in a related proof-of-principle implementation (20) ).
78
ClvR
has two components. The first is a DNA sequence modifying enzyme such as Cas9 and one
79
or more gRNAs. These constitute the
Cleaver
, are expressed in the germline and act in
trans
to
80
disrupt the endogenous version of an essential gene, creating potentially lethal LOF alleles in the
81
germline, and in the zygote due to maternal carryover of active Cas9/gRNA complexes. The
82
second is a recoded version of the essential gene resistant to cleavage that acts in
cis
to guarantee
83
the survival of those who carry it (the
Rescue
). The lethal LOF phenotype manifests itself in
84
those who fail to inherit
ClvR
and have no other functional copies of the essential gene, while
85
those who inherit
ClvR
and its associated
Rescue
survive. In this way, as with many other
86
toxin-antidote-based selfish genetic elements found in nature (reviewed in (21) ) and created de
4
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
87
novo (22) ,
ClvR
gains a relative transmission advantage that can drive it to transgene or allele
88
fixation by causing the death of those who lack it (18–20, 23) . Importantly, once a
ClvR
element
89
has spread to transgene fixation (and unlike other selfish elements in Nature), all endogenous
90
wild-type alleles of the essential gene have been eliminated through cleavage and LOF allele
91
creation. At this point the only source of essential gene function comes from
ClvR
itself––a form
92
of genetic addiction––creating a state of permanent transgene fixation. In consequence, if
93
function of the
Rescue
, a kind of Trojan horse now omnipresent in a population, is
94
condition-dependent, so too will be the survival of that population.
95
96
One environmental cue that could in principle be used to bring about conditional lethality
97
associated with a population crash is seasonal temperature.
Drosophila suzukii
, an invasive
98
species of Europe, Asia and North and South America (24, 25) , is one potential target for such an
99
approach. It has a number of generations per year and is often invasive in temperate climates that
100
experience large seasonal temperature variations (26) , providing opportunities for introducing a
101
temperature-dependent population bottleneck as a method of suppression. As a
102
proof-of-principle demonstration of this idea we sought to create a version of
ClvR
in
Drosophila
103
melanogaster
in which
Rescue
function is temperature sensitive (TS; TS
-ClvR
). We show that a
104
TS-
ClvR
element can successfully spread a conditional
Rescue
into
Drosophila
populations.
105
When populations now dependent on this transgene are shifted to non-permissive temperatures,
106
they rapidly become sterile and go extinct.
107
5
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
108
Results
109
Insertion of a TS-intein into the
Drosophila
essential gene
dribble
(
dbe
) results in
110
temperature-sensitive loss of function.
Traditional approaches to generation of dominant or
111
recessive TS mutations in essential genes in metazoans are laborious as they involve random
112
mutagenesis of whole genomes followed by large-scale screens at different temperatures for
113
otherwise fit TS mutants. As an alternative we sought to create TS versions of an essential gene
114
by introducing a TS version of an intein into the protein coding sequences of
Rescue
transgenes
115
within
ClvR
s previously shown to spread into wildtype populations (Fig. 1 and (18, 19) ). An
116
intein is a protein-encoded autoprocessing domain able to excise itself from a polypeptide and
117
rejoin the N-and C-terminal flanking sequences (exteins) to create a WT version of the encoded
118
protein (27) . Importantly, once an intein has been introduced into the coding sequence of an
119
essential gene and that version provides the only source of essential gene function, splicing
120
activity cannot be lost through mutation since the non-spliced version is non-functional.
121
122
The
Sce
VMA intein, which is located within the
Saccharomyces cerevisiae
vacuolar membrane
123
ATPase, is able to excise itself from a number of foreign proteins (28) . TS versions of
Sce
VMA
124
inteins have been isolated that allow spicing at a range of low, but not higher temperatures
125
(ranging from 18
O
C to 30
O
C (29, 30) ). A mechanistic requirement for successful intein splicing
126
is that the C-terminal extein starts with a cysteine residue. Other less well characterized sequence
127
contexts also regulate splicing efficiency (31–33) . To determine if
ClvR
Rescue
genes that
128
contain the
Sce
VMA intein are functional we generated twelve WT- and TS-intein-bearing
6
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
129
versions of
Rescue
transgenes for two previously described
ClvR
target genes, (
dribble
[
dbe
],
in
130
ClvR
dbe
(19) and
technical knockout
[
tko
], in
ClvR
tko
(18)
,
Fig. S1). We tested the ability of
131
intein-bearing
Rescue
transgenes to provide essential gene function by examining progeny of a
132
cross between females heterozygous for complete
ClvR
dbe
or
ClvR
tko
elements and males
133
heterozygous for the corresponding WT-intein
Rescue
(
Rescue
-INT
WT
) or TS-intein
Rescue
134
(
Rescue-
INT
TS
) transgene.
135
136
Fig. 1. TS-
ClvR
design and concept. (A)
TS
-ClvR
drive element comprised of Cas9/gRNAs targeting an essential
137
gene and a recoded
Rescue
of that gene with a TS
-
intein within its coding region. After translation the TS-intein can
138
splice itself out to yield a functional
Rescue
protein.
(B) Population suppression with a TS-
ClvR
.
TS
-ClvR
bearing
139
flies (red) are released into a WT population (yellow). The TS-
ClvR
selfish element spreads into the population at
140
the cost of WT. Once the TS
-ClvR
element has reached genotype fixation (has at least one Copy of TS-
ClvR
) in the
141
population, all functional endogenous copy of the essential gene targeted by TS
-ClvR
will have been mutated to
142
LOF. At this point the
conditional
TS
-Rescue
within the
ClvR
element provides the only source of essential gene
143
function in the population, making it subject to a collapse in response to a temperature shift.
144
145
When present in females,
ClvR
dbe
and
ClvR
tko
cleave and create LOF alleles of their target genes
146
in the maternal germline and the zygote with a frequency of >99.9%. Thus, in the absence of
147
another source of
Rescue
activity essentially all viable progeny should be
ClvR
-bearing (in an
7
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
148
outcross the 50% that fail to inherit
ClvR
die because they lack a functional copy of the essential
149
gene). In contrast, if the
Rescue
-INT
WT
or
Rescue
-INT
TS
in heterozygous males is active, ~33%
150
of viable progeny should be non-
ClvR
-bearing, and these should all carry the intein-bearing
151
Rescue
. From crosses carried out at 23
o
C and 27
o
C we identified one version of the
dbe Rescue
152
that retained function, in which the intein was inserted N-terminal to cysteine 2 of the
dbe
coding
153
sequence (Table S1 and S2). The
dbe
Rescue
transgene carrying the WT-intein was functional at
154
23
o
C and 27
o
C. The
Rescue
carrying the TS-intein was also functional at 23
o
C but was largely
155
(though not completely) non-functional at 27
o
C (see Fig. 2 and Table S2). Flies carrying the
dbe
156
Rescue-
INT
TS
construct were then used as a genetic background in which to create flies carrying
157
a full
ClvR
dbe
-INT
TS
(referred to as TS-
ClvR
dbe
)
drive element carrying the other components
158
found in
ClvR
dbe
(19) . These include Cas9 expressed under the control of the germline regulatory
159
sequences from the
nanos
gene, four gRNAs targeting the endogenous
dbe
locus expressed under
160
the control of individual U6 promoters, and an
OpIE
-
td-tomato
marker gene (Fig. S1B,C).
161
162
TS
-ClvR
dbe
efficiently creates LOF alleles at permissive temperatures.
A TS-
ClvR
must be
163
able to efficiently create LOF alleles at all relevant environmental temperatures, and Cas9
164
activity has been shown to be temperature sensitive, with reduced activity at lower temperatures
165
(34, 35) . To test the ability of Cas9 to create
dbe
LOF alleles at temperatures permissive for
166
intein splicing we crossed heterozygous TS-
ClvR
dbe
females to
w
1118
(WT) males at 22
o
C and
167
scored viable progeny for inheritance of the TS
-ClvR
dbe
marker. As discussed above, if the
168
TS-
ClvR
dbe
Cas9/gRNAs successfully create
dbe
LOF alleles in the maternal germline and in the
169
early embryo, viable progeny should be largely or exclusively TS-
ClvR
dbe
-bearing.
ClvR
was
8
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
170
present in 93.8% of the offspring, a lower frequency than previously reported for the original
171
ClvR
dbe
(>99% (19) ), in which crosses were carried out at 26
o
C. This is likely due to reduced
172
Cas9 activity since similar tests with the original
ClvR
dbe
stock at 22
o
C also resulted in a reduced
173
drive inheritance of 95.9% (Table S3). In any case, the results of crosses, and sequencing of
174
genomic DNA of escapers from the above crosses, show that the modestly reduced rate of
175
cleavage was not associated with the creation of functional, cleavage resistant alleles (Data S1).
176
177
Female TS-
ClvR
dbe
flies suffer a temperature-dependent loss of reproductive output.
In
178
order to bring about condition-dependent population suppression following gene drive-based
179
population modification, carriers must experience a high fitness cost under non-permissive
180
conditions. A major determinant of fitness is reproductive output, which requires ongoing adult
181
germline and somatic cell proliferation and growth.
Dbe
is a gene whose product is required in
182
all proliferative cells (36) . Thus, reproductive output is likely to be a sensitive indicator of
dbe
183
function and the effects of dosage at different temperatures. To explore these topics, we
184
characterized the reproductive output of females having two, one or no copies of TS-
ClvR
dbe
. We
185
focused on females because adult sexual maturation requires cell proliferation and growth of
186
somatic and germline cells. In contrast, young adult males already contain large numbers of
187
mature sperm, which have a long functional lifetime once deposited in the female reproductive
188
tract (37) . For each cross, four replicate vials having 5 females and 5 males (derived from flies
189
raised at 22
o
C) were incubated at different temperatures ranging from 23
o
C to 29
o
C, and
190
transferred to fresh vials every two days. The cumulative adult fly output from these crosses over
191
time is plotted in Fig. 2 (see also Fig. S2). At the low temperature of 23
o
C, crosses between
9
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
192
homozygous WT (
w
1118
) flies resulted in the production of progeny at a roughly constant rate,
193
with only a modest drop off in production during days 10-12. The rate of offspring production
194
over time was similar for crosses involving homozygous (non-TS)
ClvR
dbe
males and females,
195
and for crosses between WT females and homozygous TS-
ClvR
dbe
males (both
ClvR
s were
196
created in a
w
1118
genetic background). In contrast, crosses between heterozygous TS-
ClvR
dbe
197
females and WT males produced fewer absolute numbers of progeny. This is expected since the
198
~50% of progeny that fail to inherit TS-
ClvR
dbe
die due to lack of essential gene function. More
199
importantly, the rate of offspring production also decreased significantly over time, suggesting
200
that in an otherwise LOF background, even at permissive temperatures, one maternal copy of the
201
dbe
Rescue
INT
TS
results in gradual loss of
dbe
-dependent maternal germline activity required for
202
reproduction.
203
At higher temperatures (25
o
C-27
o
C) the loss of reproductive potential of TS-
ClvR
dbe
-bearing
204
adult females as compared to WT or those carrying
ClvR
dbe
was more dramatic. At 29
o
C
205
heterozygous TS-
ClvR
dbe
females became sterile immediately, while homozygous TS-
ClvR
dbe
flies
206
became sterile after 2 days. Progeny production also ended somewhat prematurely at 29
o
C for
207
crosses in which the female parent was WT or
ClvR
dbe
-bearing. However, this appears to be a
208
general temperature effect since the ability to produce progeny was lost at a similar rate for both
209
sets of crosses. These results, along with those described above involving crosses of
ClvR
dbe
/+
210
females to
dbe
Rescue
INT
TS
males at different temperatures, and data presented in Tables S3 and
211
S4, show that females carrying TS-
ClvR
dbe
(the vast majority of which lack
dbe
function from the
212
endogenous locus in the germline and early embryo; Table S3) are reproductively fit at lower
213
temperature, but rapidly lose the ability to reproduce at elevated temperatures.
10
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
214
215
Fig. 2:
Cumulative adult fly output at different temperatures.
Shown is the cumulative adult progeny output of
216
four replicates in which 5 females were crossed to 5 males over 12 days. Crosses were heterozygous ♀
TS-ClvR
dbe
/+
217
XX ♂w
1118
in red, homozygous ♀
TS-ClvR
dbe
/TS-ClvR
dbe
XX ♂TS-ClvR
dbe
/TS-ClvR
dbe
in yellow, ♀
w
1118
XX
218
♂TS-ClvR
dbe
/TS-ClvR
dbe
in violet, ♀
w
1118
XX ♂w
1118
(control) in blue,
and the original non-
TS
♀
ClvR
dbe
XX
♂ClvR
dbe
219
(control) in green.
220
221
TS-
ClvR
dbe
spreads to transgene fixation at a permissive temperature.
Population
222
modification followed by suppression requires that drive into a WT population succeed at low,
223
permissive temperatures. To test the ability of TS-
ClvR
dbe
to achieve this end we carried out a
224
gene drive experiment at 22
o
C. To seed the drive, we crossed heterozygous TS
-ClvR
dbe
males
225
(
w
1118
; TS-
ClvR
dbe
/+) to WT (
w
1118
) females to create a starting TS-
ClvR
dbe
allele frequency of
11
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
226
25%, in four replicate populations. Mated females were allowed to lay eggs in a food bottle for
227
one day and removed afterwards. The drive experiments were kept in a temperature-controlled
228
incubator at 22
o
C. After ~16 days most progeny had developed into adults, which were then
229
removed from the bottles, scored for the presence of the TS-
ClvR
dbe
marker (
td-tomato
), and
230
transferred to a fresh food bottle to repeat the cycle. Results of the drive experiment are shown in
231
Fig. 3A. The TS-
ClvR
dbe
construct reached genotype fixation between 9 and 10 generations in all
232
4 replicate drive populations, while a construct carrying only the
dbe
Rescue-
INT
TS
but no
233
Cas9/gRNAs did not increase in frequency. By generation 18 TS-
ClvR
dbe
allele frequencies
234
ranged from 93.2-97.6% (Table S5).
235
236
Fig. 3. Population modification at a permissive temperature followed by suppression at a restrictive
237
temperature. (A)
Shown are genotype frequencies of TS-
ClvR
dbe
-bearing flies over discrete generations at 22
o
C.
238
TS-
ClvR
dbe
is indicated with solid lines,
dbe
Rescue-
INT
TS
controls with dashed lines.
(B)
Gray lines show individual
239
population trajectories for all replicates when incubated at 29
0
C. All populations produced some offspring when
240
moved from 22
o
C to 29
o
C. These collapsed in the next generation due to complete sterility.
241
242
Populations in which TS
-ClvR
dbe
is ubiquitous undergo a population collapse when shifted
12
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
243
to elevated temperature.
The goal of drive with a TS-
ClvR
is ultimately to bring about a
244
population crash in response to an environmental temperature shift once LOF allele creation
245
associated with population modification has rendered all members of the population dependent
246
on the
Rescue
-INT
TS
. As a test of this hypothesis, we followed the fate of drive populations
247
shifted to 29
o
C at generations 10, 12, 13, 16 and 17. At each of these points adults from the 22
o
C
248
drive population were allowed to lay eggs for one day at 22
o
C in order to continue the drive, and
249
then moved to 29
o
C to allow egg laying for a further two days. Adults were then removed and
250
the fate of the 29
o
C populations followed, as with the drive populations kept at 22
o
C (Table S6).
251
Populations fixed for
ClvR
dbe
(control) individuals produce many adult progeny over 6
252
generations when continuously housed at 29
o
C (c.f. Table S7). In contrast, populations of drive
253
individuals–which at this point are heterozygous or homozygous for TS-
ClvR
dbe
–give rise to only
254
a few adult progeny per parent for one more generation (c.f. gray line leading from the number
255
of generation 10 individuals transferred to 29
o
C to the generation 11 adult progeny number).
256
These latter adults were universally sterile, resulting in population extinction in the next
257
generation (Fig. 3D).
258
259
DISCUSSION
260
Our results show that gene drive can be used to spread a trait conferring conditional lethality into
261
an insect population, resulting in a population crash when the restrictive condition, in this case a
262
temperature shift, is experienced. Additional Cargo genes, designed to bring about some other
263
phenotype such as disease suppression prior to temperature-dependent population suppression
13
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
264
could also be included in such gene drive elements. The implementation described herein used
265
the
ClvR
gene drive mechanism, which concurrently renders LOF endogenous copies of an
266
essential gene and replaces them with a TS version as spread occurs. A similar outcome (drive
267
followed by condition-dependent suppression) could also be achieved using strategies in which a
268
HEG homes into an essential gene locus, thereby disrupting its function, while also carrying a
269
cleavage-resistant version of the essential gene as a rescuing transgene (38–42) , that in this case
270
is engineered to be temperature sensitive.
271
Conditional populations suppression systems target both males and females when a
272
sex-independent essential gene is utilized for cleavage and conditional rescue, as described here.
273
With such a system the target environment may require some level of periodic repopulation with
274
transgenes. A modified system that would reduce this need, and work to maintain the transgene
275
in the target environment in the face of incoming migration of WT, eliminates only females or
276
female fertility under non-permissive conditions (for modeling of a related system with these
277
characteristics see (43) ).
ClvR
s that bring about LOF and
Rescue
of two different genes, one that
278
is needed for sex-independent viability (mediating strong drive) and a second that is required for
279
female viability or fertility (allowing for elimination of females under non-permissive
280
conditions), could be used to achieve this goal.
ClvR
s able to rescue the viability and fertility
281
associated with LOF of two different essential genes at the same time have been created (18,
282
19) ), suggesting this approach is plausible. Finally, we note that the strategy for generating TS
283
strains described here (replacement of a WT version of an endogenous gene with a TS-version)
284
could also be used as a method of sex-specific sorting in inundative suppression strategies such
14
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
285
as the sterile insect technique.
286
287
Success with any TS gene drive system in the wild will require knowledge of temperature
288
fluctuations within a season in the region of interest, the life phases in which the target species is
289
most susceptible (and resistant) to loss of essential gene function, and potentially further
290
selections in rapidly reproducing organisms like yeast (29, 30) for TS-inteins best suited to the
291
environmental temperature regimes involved. Also, because seasonal temperatures do not change
292
in an all or none fashion, gradual shifts towards non-permissive conditions will provide
293
opportunities for selection to take place on sequences within the intein coding region that reduce
294
or eliminate temperature sensitivity. The targeting of biosynthetic essential genes such as
dbe
,
295
whose transient LOF is unlikely to result in an immediate fitness cost (as is seen for some other
296
TS mutants that cause immediate paralysis; c.f. (44) ) probably provides some level of
297
environmental phenotypic buffering in this regard but would not eliminate selection. While next
298
generation
ClvR
elements can be cycled through a population, replacing old, failed elements with
299
new ones (19) , strategies that forestall the need for such cycles of modification for as long as
300
possible would be useful. This can be achieved by building into the
Rescue
transgene
301
mechanistic redundancy with respect to how temperature sensitivity is achieved, thereby
302
necessitating multiple mutational hits for the
Rescue
to lose its TS characteristic. As an example,
303
an N-terminal TS degron (the N-terminal location preventing the loss of degron activity through
304
frameshift or stop codons) that promotes the degradation of a linked C-terminal protein at
305
elevated temperature provides one such approach (45) . Insertion of multiple copies of a common
15
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
306
TS intein at different positions provides another.
307
308
Finally, we note that a similar logic to that presented here, in which
Rescue
activity is
309
conditionally blocked, could be used to bring about species-specific suppression in response to
310
other stimuli. Small molecules provide one example. These could block intein splicing activity
311
(46) , promote the degradation of a target protein (47) , or decrease the stability of specific
312
transcripts (48) . Target genes that might be particularly amenable to such approaches, which will
313
likely alter expression only transiently following application, include those encoding proteins
314
whose loss results in rapid cell death, such as inhibitors of apoptosis (49) . Virus infection
315
provides a further opportunity for engineering conditional lethality. As an example,
316
virus-encoded protease activity, required for viral polyprotein processing in many systems,
317
serves as an “honest” and specific indicator of infection. If one or more viral protease target sites
318
are engineered into the products of key host essential genes–-and these versions replace WT
319
counterparts during drive–-cleavage at these sites in organisms that are virally infected could
320
result in a lethal LOF phenotype. This could be used to directly suppress populations in response
321
to introduction of a naturally-occurring and otherwise benign virus. A similar strategy could also
322
be used to selectively eliminate members of a disease vector population that are infected with a
323
human, animal or plant pathogenic virus, in the context of a simple population modification
324
scenario.
325
Acknowledgments:
Stocks obtained from the Bloomington Drosophila Stock Center (NIH
326
P40OD018537) were used in this study.
16
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
327
Funding:
This work was carried out with support to BAH from the US Department of
328
Agriculture, National Institute of Food and Agriculture specialty crop initiative under USDA
329
NIFA Award No. 2012-51181-20086 and the Caltech Resnick Sustainability Institute. G.O. was
330
supported
by a Baxter Foundation Endowed Senior Postdoctoral Fello wship
and the Caltech
331
Resnick Sustainability Institute. T.I. was supported by NIH training grant 5T32GM007616-39.
332
Author Contributions:
Conceptualization, G.O., T.I. and B.A.H.; Methodology, G.O., T.I. and
333
B.A.H.; Investigation, G.O. and B.A.H.; Writing – Original Draft, G.O. and B.A.H.; Writing –
334
Review & Editing, G.O., T.I. and B.A.H.; Funding Acquisition, G.O. and B.A.H.
335
Competing interests:
The authors have filed patent applications on
ClvR
and related
336
technologies (U.S. Application No.
15/970,728
and
No. 16/673,823
; provisional patent
No.
337
CIT-8511-P
).
338
Data availability:
All data is available in the main text or the supplementary materials.
339
340
References
341
1. B. A. Hay, G. Oberhofer, M. Guo, Engineering the Composition and Fate of Wild
342
Populations with Gene Drive.
Annu. Rev. Entomol.
(2020)
343
https:/doi.org/ 10.1146/annurev-ento-020117-043154 .
344
2. M. J. Whitten, Insect control by genetic manipulation of natural populations.
Science
171,
345
682–684 (1971).
346
3. A. Burt, Site-specific selfish genes as tools for the control and genetic engineering of
347
natural populations.
Proc. Biol. Sci.
270, 921–928 (2003).
348
4. L. E. LaChance, E. F. Knipling, Control of Insect Populations through Genetic
349
Manipulations.
Ann. Entomol. Soc. Am.
55, 515–520 (1962).
350
5. P. Schliekelman, F. Gould, Pest control by the introduction of a conditional lethal trait on
351
multiple loci: potential, limitations, and optimal strategies.
J. Econ. Entomol.
93,
17
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint
352
1543–1565 (2000).
353
6. A. Deredec, A. Burt, H. C. J. Godfray, The population genetics of using homing
354
endonuclease genes in vector and pest management.
Genetics
179, 2013–2026 (2008).
355
7. H. C. J. Godfray, A. North, A. Burt, How driving endonuclease genes can be used to combat
356
pests and disease vectors.
BMC Biol.
15, 81 (2017).
357
8. A. Hammond,
et al.
, A CRISPR-Cas9 gene drive system targeting female reproduction in
358
the malaria mosquito vector Anopheles gambiae.
Nat. Biotechnol.
34, 78–83 (2016).
359
9. A. M. Hammond,
et al.
, The creation and selection of mutations resistant to a gene drive
360
over multiple generations in the malaria mosquito.
PLoS Genet.
13, e1007039 (2017).
361
10. K. Kyrou,
et al.
, A CRISPR-Cas9 gene drive targeting doublesex causes complete
362
population suppression in caged Anopheles gambiae mosquitoes.
Nat. Biotechnol.
(2018)
363
https:/doi.org/ 10.1038/nbt.4245 .
364
11. R. Galizi,
et al.
, A synthetic sex ratio distortion system for the control of the human malaria
365
mosquito.
Nat. Commun.
5, 3977 (2014).
366
12. R. Galizi,
et al.
, A CRISPR-Cas9 sex-ratio distortion system for genetic control.
Sci. Rep.
6,
367
31139 (2016).
368
13. B. Fasulo,
et al.
, A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9
369
sex distorters.
PLoS Genet.
16, e1008647 (2020).
370
14. A. Simoni,
et al.
, A male-biased sex-distorter gene drive for the human malaria vector
371
Anopheles gambiae.
Nat. Biotechnol.
(2020) https:/doi.org/ 10.1038/s41587-020-0508-1 .
372
15. A. Meccariello,
et al.
, Engineered sex ratio distortion by X-shredding in the global
373
agricultural pest Ceratitis capitata.
BMC Biol.
19, 78 (2021).
374
16. M. KaramiNejadRanjbar,
et al.
, Consequences of resistance evolution in a Cas9-based sex
375
conversion-suppression gene drive for insect pest management.
Proc. Natl. Acad. Sci. U. S.
376
A.
115, 6189–6194 (2018).
377
17. F. Gould, P. Schliekelman, Population genetics of autocidal control and strain replacement.
378
Annu. Rev. Entomol.
49, 193–217 (2004).
379
18. G. Oberhofer, T. Ivy, B. A. Hay, Cleave and Rescue, a novel selfish genetic element and
380
general strategy for gene drive.
Proc. Natl. Acad. Sci. U. S. A.
(2019)
381
https:/doi.org/ 10.1073/pnas.1816928116 .
382
19. G. Oberhofer, T. Ivy, B. A. Hay, Gene drive and resilience through renewal with next
383
generation Cleave and Rescue selfish genetic elements.
Proc. Natl. Acad. Sci. U. S. A.
117,
18
.
CC-BY-NC-ND 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted July 4, 2021.
;
https://doi.org/10.1101/2021.07.03.451005
doi:
bioRxiv preprint