Supplementary Materials for
Gene drive that brings about addiction to a temperatur
e sensitive version of an essential
gene triggers a population collapse in
Drosophila
Georg Oberhofer
, Tobin Ivy
, and Bruce
A Hay*
Correspondence to: haybruce@caltech.edu
This PDF
file includes:
Fig. S1 to S3
Table S1 to S9
Other Supplementary Materials for
this manuscript include the following:
Data S1: Gene drive counts, Control drive counts, Escaper crosses, Escaper tar
get site
sequencing results, primers, synthetic constructs genbank files
0
Fig.
S1:
(A)
Genomic
insertion
of
the
R
e
s
c
u
e
-
INT
constructs.
We
assembled
plasmids
that
had
TS
and
WT
versions
of
the
VMA
intein
inserted
into
the
coding
regions
of
dbe
and
tko
.
The
constructs
also
had
a
ubiquitous
OpIE
-GFP
marker,
and
an
attP
landing
site
for
subsequent
modifications
of
the
locus.
These
were
flanked
by
homology
arms
to
facilitate
CRISPR-HR
mediated
insertion
into
the
genome.
The
construct
was
injected
into
w
1118
flies
along
with
a
Cas9
RNP
complex
that
targeted
the
genomic
region
at
68E
on
the
third
chromosome.
(B)
Genomic
integration
of
Cas9/gRNAs.
The
second
part
of
the
ClvR
drive
mechanism,
Cas9
and
the
gRNAs,
were
integrated
into
the
genomic
site
of
the
TS-
Rescue
to
yield
complete
TS
-ClvR
dbe
flies.
This
second
step
was
performed
only
with
flies
carrying
the
INT
TS
(
dbe
)Cys2.
(C)
Schematic
of
the
final
TS
-
C
l
v
R
dbe
drive
element.
(D)
Intein insertion positions in
d
b
e
and
t
k
o
coding regions
1
Fig.
S2:
Identification
and
characterization
of
"escapers",
progeny
of
ClvR
-bearing
females
that
survive
and
lack
ClvR
.
2
Fig.
S3:
Adult
fly
output
at
different
temperatures.
Shown
are
the
numbers
of
adult
flies
in
four
replicates
that
eclosed
from
different
crosses
incubated
at
23
o
C
(A)
,
25
o
C
(B)
,
27
o
C
(C)
,
and
29
o
C
(D)
over
12
days
of
egg-laying.
Crosses
were
♀TS
-ClvR
dbe
/+
X
♂w
1118
in
red,
♀TS-
ClvR
dbe
/
TS
-ClvR
dbe
X
♂
TS
-ClvR
dbe
/
TS
-ClvR
dbe
in
yellow,
♀
w
1118
X
♂
TS
-ClvR
dbe
/TS
-ClvR
dbe
in
violet,
♀
ClvR
dbe
X
♂ClvR
dbe
(control)
in
green,
and
♀
w
1118
X
♂w
1118
(control)
in
blue.
Cumulative sums of adult progeny are shown in Fig. 3 in the main text.
3
Supplementary
Tables
Table
S1:
Screening
of
R
e
s
c
u
e
-INT
function
for
t
k
o
.
Shown
are
the
numbers
of
offspring
from
single
fly
crosses
of
heterozygous
female
ClvR
tko
/+
to
males
that
carry
a
copy
of
different
versions
of
the
Rescue-
INT
for
tko
.
Crosses
were
kept
at
23
o
C
or
27
o
C.
WT
(+/+)
offspring
were
dying
from
maternal
carryover
activity
of
ClvR
tko
at
both
temperatures.
Offspring
that
carry
the
Rescue
within
ClvR
tko
were
not
affected
by
temperature.
Neither
INT
WT
nor
INT
TS
versions
of
the
tko
Rescue
were
able
to
rescue
the
LOF
phenotypes
induced
by
the
ClvR
tko
element.
Note:
We
did
not
obtain
transformants
for
the
INT
TS
version
inserted
after
cysteine
2
of
tko
.
Since
the
INT
WT
version
of
that
Rescue
did
not
provide
gene
function
we
reasoned
that
the
TS-version
will
not
work
either.
Thus,
the
construct
was
not pursued further.
Rescue
Temperature
Replicate
R
e
s
c
u
e
/
+
Rescue/ClvR
ClvR/+
+/+
notes
INT
TS
(tko)Cys1
23
A
0
41
46
0
23
B
0
42
53
0
23
C
0
50
55
0
27
A
0
49
49
0
27
B
0
51
59
0
27
C
0
44
41
0
INT
TS
(tko)Cys3
23
A
0
45
43
0
23
B
0
53
58
0
23
C
0
41
39
0
27
A
0
44
46
0
27
B
0
60
53
0
27
C
0
41
36
0
INT
WT
(tko)Cys1
23
A
0
58
48
0
23
B
0
54
52
0
23
C
0
55
48
0
27
A
0
44
48
0
27
B
0
41
40
0
27
C
-
-
-
-
sterile
INT
WT
(tko)Cys2
23
A
0
48
44
0
23
B
1
42
45
0
23
C
0
53
60
0
27
A
0
52
57
0
27
B
0
40
45
0
27
C
0
62
60
0
INT
WT
(tko)Cys3
23
A
1
40
42
0
23
B
0
50
46
0
23
C
0
45
44
0
27
A
0
47
47
0
27
B
0
36
40
0
27
C
0
63
62
0
4
Table
S2.
Screening
of
intein-
R
e
s
c
u
e
function
for
d
b
e
.
Shown
are
the
numbers
of
adult
offspring
output
from
single
fly
crosses
of
heterozygous
female
ClvR
dbe
/+
to
males
that
carry
a
copy
of
different
versions
of
the
Rescue
-INT
for
dbe
.
Crosses
were
kept
at
23
o
C
or
27
o
C.
WT
(+/+)
offspring
of
ClvR
dbe
mothers
die
due
to
LOF
allele
creation
in
the
female
germline
and
zygote
at
both
temperatures.
Offspring
that
carry
the
Rescue
within
ClvR
dbe
were
not
affected
by
temperature.
Versions
with
a
INT
WT
inserted
after
cysteine
2
or
3
of
dbe
were
functional
at
both
temperatures.
Versions
with
a
INT
TS
inserted
after
cysteine
and
1
and
3
did
not
provide
Rescue
function
at
either
temperature.
However,
a
INT
TS
inserted
after
cysteine
2
showed
promising
behavior,
having
Rescue
activity
at
23
o
C,
whereas
at
27
o
C
most
of
the
flies
that
carried
it
did
not
develop
into
adults
(highlighted
in
red).
We
chose
this
Rescue
-INT
TS
to
build
a
full
TS
-ClvR
element
by
inserting
Cas9
and
gRNAs
from
ClvR
dbe
.
Note:
For
the
INT
WT
inserted
after
cysteine
1
we
did
not
obtain
transformants
after
a
first
round
of
injections.
Since
the
INT
TS
version
inserted
after
cysteine
1
did
not show any
Rescue
activity we did not pursue this
construct further.
Rescue
Temperature
Replicate
R
e
s
c
u
e
/
+
Rescue/ClvR
ClvR/+
+/+
INT
TS
(dbe)Cys1
23
A
0
35
39
0
23
B
0
35
32
0
23
C
0
31
36
0
27
A
0
40
45
0
27
B
0
39
32
0
27
C
0
47
50
0
INT
TS
(dbe)Cys2
23
A
20
26
23
0
23
B
31
24
24
2
23
C
30
32
36
0
27
A
3
25
23
0
27
B
4
37
39
0
27
C
3
28
24
0
INT
TS
(dbe)Cys3
23
A
0
49
49
0
23
B
0
33
35
0
23
C
0
36
32
0
27
A
0
44
47
0
27
B
0
38
37
0
27
C
0
32
30
0
INT
WT
(dbe)Cys2
23
A
47
45
54
0
23
B
41
30
25
0
23
C
48
46
43
0
27
A
41
33
26
0
27
B
29
23
31
0
27
C
26
41
43
0
INT
WT
(dbe)Cys3
23
A
47
43
52
0
23
B
20
52
40
0
23
C
6
51
36
0
27
A
41
33
26
0
27
B
29
23
31
0
27
C
26
41
43
0
5
Table
S3:
Cleavage
to
LOF
of
C
l
v
R
dbe
and
TS
-
C
l
v
R
dbe
at
22
o
C.
We
assayed
the
cleavage
activity
of
TS
-ClvR
dbe
at
the
permissive
temperature
of
22
o
C
by
crossing
heterozygous
TS
-ClvR
dbe
females
to
w
1118
males
and
scoring
the
offspring
for
the
dominant
td-tomato
marker.
The
observed
frequency
of
TS
-ClvR
-bearing
flies
in
the
offspring
was
lower
than
what
we
previously
observed
with
ClvR
dbe
(>99%,
(27)
).
That
experiment
was
performed
at
a
higher
temperature
of
26
o
C.
Since
the
cleaving
components
(Cas9/gRNAs)
of
TS
-ClvR
dbe
are
exactly
the
same
as
for
ClvR
dbe
we
reasoned
that
the
lower
cleavage
activity
might
be
due
to
the
lower
incubation
temperature.
To
confirm
this,
we
set
up
the
same
crosses
with
the
original
ClvR
dbe
stock
incubated
at
22
o
C
and
found
a
lower
rate
of
cleavage
to
LOF
in that stock as well.
Control crosses
♀
C
l
v
R
dbe
/
+
X ♂
w
1
1
18
Bottle
ClvR
-bearing
♂
w
1118
♀
w
1118
sum
ClvR
-freq (%)
cleavage to LOF (%)
A
817
17
14
848
96.34
92.69
B
800
21
19
840
95.24
90.48
C
831
16
13
860
96.63
93.26
D
597
18
14
629
94.91
89.83
total
3045
72
60
3177
95.85
91.69
Crosses with TS-
C
l
v
R
dbe
/
+
X
w
1
1
18
Bottle
ClvR
-bearing
♂
w
1118
♀
w
1118
sum
ClvR
-freq (%)
cleavage to LOF (%)
A
832
22
24
878
94.76
89.52
B
975
31
37
1043
93.48
86.96
C
385
14
10
409
94.13
88.26
D
575
24
22
621
92.59
85.19
total
2767
91
93
2951
93.76
87.53
6
Table
S4:
Incubations
at
a
restrictive
temperature
of
29
o
C.
In
a
first
test
we
crossed
homozygous
♀TS
-ClvR
dbe
to
♂
TS
-ClvR
dbe
and
incubated
them
at
a
potentially
restrictive
temperature
of
29
o
C.
We
also
set
up
controls
with
w
1118
X
w
1118
and
homozygous
ClvR
dbe
X
ClvR
dbe
.
All
flies
were
reared
at
22
o
C,
crossed
to
each
other
in
a
fresh
food
vial
and
transferred
to
a
29
o
C
incubator.
All
the
crosses
were
fertile
and
gave
progeny
in
the
F1
generation.
We
transferred
all
the
F1
flies
to
a
fresh
vial
and
kept
them
at
29
o
C.
F1
progeny
of
TS
-ClvR
dbe
X
TS
-ClvR
dbe
were
completely
sterile,
whereas
F1
progeny
from
the
two
control
crosses
remained
fertile.
F1
progeny
of
all
crosses
were
monitored
for
1
week
at
29
o
C.
Afterwards
we
took
two
male
TS-
ClvR
dbe
flies
and
crossed
them
to
w
1118
virgins.
We
also
took
two
females
and
crossed
them
to
w
1118
males.
Both
crosses
did
not
yield
offspring.
The
remaining
F1
flies
of
the
TS
-ClvR
dbe
cross
were
put
back
at
22
o
C
and
monitored
for
another
week
after
which
most
of
them
had
died.
All
of
the
flies remained sterile.
Cross
Vial
F1
F2
w
1118
X
w
1118
1
66
fertile
2
85
fertile
3
102
fertile
4
110
fertile
5
95
fertile
ClvR
dbe
X
ClvR
dbe
1
95
fertile
2
99
fertile
3
98
fertile
4
103
fertile
5
106
fertile
TS
-ClvR
dbe
X TS
-ClvR
dbe
1
32
sterile
2
60
sterile
3
63
sterile
4
25
sterile
5
18
sterile
6
64
sterile
7
69
sterile
8
67
sterile
9
63
sterile
10
83
sterile
7
Table S5: Male
T
S
-
C
l
v
R
dbe
and
w
1
1
18
(control) fertility
at 23
o
C.
TS-
ClvR
dbe
(
w
1118
as controls) adult males
raised at
23
o
C were crossed to four
w
1118
virgins in a vial
and incubated at a restrictive temperature of 29
o
C
and a permissive
temperature of 23
o
C. Every two days we transferred
the males to a fresh food vial and added new virgins.
♂
TS-ClvR
dbe
X ♀
w
1118
@ 23
o
C
♂
w
1118
X ♀
w
1118
@ 23
o
C
Time
(days)
Replicate
Fertility
Time (days)
Replicate
Fertility
0-2
A
fertile
0-2
A
fertile
0-2
B
fertile
0-2
B
fertile
0-2
C
fertile
0-2
C
fertile
0-2
D
fertile
0-2
D
fertile
0-2
E
fertile
0-2
E
fertile
2-4
A
fertile
2-4
A
fertile
2-4
B
fertile
2-4
B
fertile
2-4
C
fertile
2-4
C
fertile
2-4
D
fertile
2-4
D
fertile
2-4
E
fertile
2-4
E
fertile
4-6
A
fertile
4-6
A
fertile
4-6
B
fertile
4-6
B
fertile
4-6
C
fertile
4-6
C
fertile
4-6
D
fertile
4-6
D
fertile
4-6
E
fertile
4-6
E
fertile
6-8
A
fertile
6-8
A
fertile
6-8
B
fertile
6-8
B
fertile
6-8
C
fertile
6-8
C
fertile
6-8
D
fertile
6-8
D
fertile
6-8
E
fertile
6-8
E
fertile
8-10
A
fertile
8-10
A
fertile
8-10
B
fertile
8-10
B
fertile
8-10
C
fertile
8-10
C
fertile
8-10
D
fertile
8-10
D
fertile
8-10
E
fertile
8-10
E
fertile
8