Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi

Abstract

Significance
Malaria continues to impose enormous health and economic burdens on the developing world. Novel technologies proposed to reduce the impact of the disease include the introgression of parasite-resistance genes into mosquito populations, thereby modifying the ability of the vector to transmit the pathogens. Such genes have been developed for the human malaria parasite Plasmodium falciparum . Here we provide evidence for a highly efficient gene-drive system that can spread these antimalarial genes into a target vector population. This system exploits the nuclease activity and target-site specificity of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, which, when restricted to the germ line, copies a genetic element from one chromosome to its homolog with ≥98% efficiency while maintaining the transcriptional activity of the genes being introgressed.

Full text

Highly efficient Cas9-mediated gene drive for
population modification of the malaria vector
mosquito Anopheles stephensi
Valentino M. Gantz
a,1
, Nijole Jasinskiene
b,1
, Olga Tatarenkova
b
, Aniko Fazekas
b
, Vanessa M. Macias
b
, Ethan Bier
a,2
,
and Anthony A. James
b,c,2
a
Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0349;
b
Department of Molecular Biology and
Biochemistry, University of California, Irvine, CA 92697-3900; and
c
Department of Microbiology and Molecular Genetics, School of Medicine, University of
California, Irvine, CA 92697-4500
Contributed by Anthony A. James, October 26, 2015 (sent for review October 11, 2015; reviewed by Malcolm Fraser and Marcelo Jacobs-Lorena)
Genetic engineering technologies can be used both to create
transgenic mosquitoes carrying antipathogen effector genes tar-
geting human malaria parasites and to generate gene-drive systems
capable of introgressing the genes throughout wild vector popula-
tions. We developed a highly effective autonomous Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated
protein 9 (Cas9)-mediated gene-drive system in the Asian malaria
vector Anopheles stephensi, adapted from the mutagenic chain re-
action (MCR). This specific system results in progeny of males and
females derived from transgenic males exhibiting a high frequency
of germ-line gene conversion consistent with homology-directed re-
pair (HDR). This system copies an ∼17-kb construct from its site of
insertion to its homologous chromosome in a faithful, site-specific
manner. Dual anti-Plasmodium falciparum effector genes, a marker
gene, and the autonomous gene-drive components are introgressed
into ∼99.5% of the progeny following outcrosses of transgenic
lines to wild-type mosquitoes. The effector genes remain tran-
scriptionally inducible upon blood feeding. In contrast to the effi-
cient conversion in individuals expressing Cas9 only in the germ
line, males and females derived from transgenic females, which
are expected to have drive component molecules in the egg, pro-
duce progeny with a high frequency of mutations in the targeted
genome sequence, resulting in near-Mendelian inheritance ratios
of the transgene. Such mutant alleles result presumably from non-
homologous end-joining (NHEJ) events before the segregation of
somatic and germ-line lineages early in development. These data
support the design of this system to be active strictly within the germ
line. Strains based on this technology could sustain control and elim-
ination as part of the malaria eradication agenda.
Plasmodium falciparum
|
MCR
|
eradication
|
transgenesis
|
CRISPR
Efforts in the ongoing campaign to eradicate malaria show
mixed success. The World Health Organization reports
that malaria mortality continues to decrease and estimates that
∼3.3 million lives have been saved since 2001 as a result of using
new drugs, personal protection, environmental modification, and
other measures (1–3). Although these gains are encouraging, there
were still ∼580,000 deaths globally in 2014 (3), a statistic that sup-
ports the continued application of proven existing control and
treatment methods while highlighting the pressing need for
strategic development and deployment of new tools.
Prevention of parasite transmission by vector mosquitoes has
always played a major role in malaria control (4, 5). However,
the challenges of vector control mirror those of malaria eradi-
cation in general and include the heterogeneity and complexity
of transmission dynamics and the difficulties in sustaining control
practices (6, 7). Genetic approaches that result in altering vector
populations in such a way as to eliminate their ability to transmit
parasites to humans (population modification) can contribute to
sustainable control and elimination by providing barriers to parasite
and competent vector reintroduction, and allow resources to be
directed to new sites while providing confidence that treated
areas will remain malaria-free (5, 7).
We and others are pursuing a population-modification ap-
proach that involves the introduction of genes that confer a par-
asite-resistance phenotype to mosquitoes that otherwise would be
fully capable of transmitting the pathogens (8–13). The expecta-
tion is that the introgression of such an effector gene at a high
enough frequency in a vector population would decrease or
eliminate transmission and result in measurable impacts on mor-
bidity and mortality (14). Critical to this approach are the devel-
opment of a gene that confers resistance to the transmission of the
parasites, transgenesis tools for introducing the genes into mos-
quito strains, and a mechanism to spread the genes at epidemio-
logically significant rates into the target populations. Working with
Anopheles stephensi, a vector of malaria in the Indian subcontinent
(15), we now have demonstrated proof of principle for all of
these components.
An. stephensi is both an established and emerging malaria
vector. It is estimated to be responsible for ∼12% of all transmission
in India, mostly in urban settings, accounting for a total of ∼106,000
clinical cases in 2014 (3, 16–18), and also may be responsible for
recent epidemic outbreaks in Africa (19). Laboratory strains of An.
Significance
Malaria continues to impose enormous health and economic
burdens on the developing world. Novel technologies pro-
posed to reduce the impact of the disease include the introgression
of parasite-resistance genes into mosquito populations, thereby
modifying the ability of the vector to transmit the pathogens.
Such genes have been developed for the human malaria par-
asite Plasmodium falciparum. Here we provide evidence for a
highly efficient gene-drive system that can spread these anti-
malarial genes into a target vector population. This system
exploits the nuclease activity and target-site specificity of the
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) system, which, when restricted to the germ line,
copies a genetic element from one chromosome to its homolog
with ≥98% efficiency while maintaining the transcriptional
activity of the genes being introgressed.
Author contributions: V.M.G., N.J., V.M.M., E.B., and A.A.J. designed research; V.M.G.,
N.J., O.T., A.F., and V.M.M. pe rformed research; V.M.G., N.J., O.T., A.F., V.M.M., E.B.,
and A.A.J. analyzed data; and V.M.G., N.J., V.M.M., E.B., and A.A.J. wrote the paper.
Reviewers: M.F., University of Notre Dame; and M.J.-L., Johns Hopkins School of Public Health.
Conflict of interest statement: E.B. and V.G. are authors of a patent applied for by the
University of California, San Diego that relates to the mutagenic chain reaction.
Freely available online through the PNAS open access option.
1
V.M.G. and N.J. contributed equally to this work.
2
To whom correspondence may be addressed. Email: ebier@ucsd.edu or aajames@uci.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1521077112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1521077112 PNAS Early Edition
|
1of8
APPLIED BIOLOGICAL
SCIENCES
PNAS PLUS

stephensi are transformed efficiently with transposable elements
facilitating analyses of transgene expression in diverse genomic
locations (20). Site-specific integration technologies adapted to
this species allow integrations of exogenous DNA into the
mosquito genome at locations with little or no impact on fitness
(11, 21). Furthermore, a dual antiparasite effector gene was
developed based on the single-chain antibodies (scFvs) m1C3
and m2A10 that target the human malaria parasite Plasmodium
falciparum ookinete protein Chitinase 1 and the circumsporozoite
protein (CSP), respectively (10, 22, 23). Transgenic An. stephensi
adult females expressing m1C3 and m2A10 had no P. falciparum
sporozoites (the infectious stage of these parasites) in their salivary
glands under infection conditions expected in the field, and there-
fore were incapable of transmitting parasites (11).
Research on mechanisms for introducing antipathogen effector
genes into target populations supports a number of approaches,
including inundative releases and those based on gene-drive sys-
tems (24). Inundative approaches rely on releases of engineered
mosquitoes in numbers substantially exceeding those of the local
population to drive gene frequencies high enough to have an epi-
demiological impact. Inundative releases of chemically or radiation-
treated insects were successful in population suppression of mos-
quitoes using sterile insect technologies (25). However, modeling of
gene-drive systems, which exceed rates of Mendelian inheritance,
shows a more rapid population-level transformation with fewer
releases than inundative approaches (24), and this would result
in sustainable local malaria elimination at much reduced costs (7).
We show here that a gene-drive system using Clustered Regu-
larly Interspaced Short Palindromic Repeats (CRISPR)-associated
protein 9 (Cas9)-mediated homology-directed repair (HDR)
adapted from a highly efficient system, mutagenic chain reaction
(MCR), developed in the fruit fly Drosophila melanogaster (26)
drives target-specific gene conversion at ≥99.5% efficiency in
transgene heterozygotes of An. stephensi. The drive system as
designed works in both the male and female germ lines of mos-
quitoes derived from transgenic males. Cas9-mediated gene
targeting also is evident in the somatic cells of embryos derived
from transgenic females. The system can carry a relatively large
set of genes (∼17 kb in length), and these are transcriptionally
active following movement. Strains based on this technology could
have a major role in sustaining malaria control and elimination
as part of the eradication agenda.
Results
Assembly, Microinjection, and Selection of Transgenic Progeny. The
structure of the gene-drive plasmid, pAsMCRkh2, is based on a
previous autocatalytically propagating element design (26) and
targets its insertion into the locus encoding the kynurenine hy-
droxylase (kynurenine monooxygenase) enzyme (Fig. 1). The
target gene is located autosomally on 3L of the An. stephensi
linkage map (15), and we refer to it as kynurenine hydroxylase
white
(kh
w
) to indicate orthology with a gene in Aedes aegypti, which
has a recessive white-eye phenotype (27–29). The pAsMCRkh2
construct has the following elements: (i)anAn. stephensi co-
don-optimized Cas9 endonuclease-encoding DNA flanked by
the putative promoter, 5′-and3′-end nucleotide sequences
of the An. stephensi vasa gene (ASTE003241), intended to
drive the expression of the nuclease in both male and female
germ lines; (ii) a putative An. stephensi U6A gene (ASTE015697)
promoter directing the expression of a guide RNA (gRNA) tar-
geting the An. stephensi kh
w
gene at a site (designated kh2) im-
mediately adjacent to two known mutations in the Ae. aegypti
orthologous gene that cause visible eye phenotypes (28, 29); (iii)a
3xP3-DsRed gene (30), which expresses the DsRed dominant
fluorescence marker visible in larval photoreceptors as well as
nonpigmented adult eyes (Fig. 2); (iv) dual antipathogen effector
genes(m2A10-m1C3)targetingP. falciparum (10, 11); and (v)
DNA fragments ∼1 kb in length each that are homologous to
the An. stephensi kh
w
locus immediately adjacent to the 5′and
3′ends of the kh2 target cut site. The resulting plasmid is a total
of ∼21 kb in length with 16,625 bp comprising the components
(“cargo”) targeted for insertion at the An. stephensi kh
w
locus.
A total of 680 G
0
wild-type embryos of the Indian strain of
An. stephensi (15) was injected with a solution containing 100 ng/μL
each of the pAsMCRkh2 plasmid, Cas9 protein, Cas9 double-
stranded RNAs (dsRNAs), and Ku70 dsRNA. The rationale for
including the dsRNAs was to silence expression of the incoming
Cas9 gene (dsCas9) carried on the plasmid and to reduce activity
of the nonhomologous end-joining (NHEJ) pathway (dsKU70)
(31) to favor HDR-mediated insertion of the pAsMCRkh2 cargo.
Genomic integration of the transgene was achieved by the coin-
jected Cas9 protein together with the kh2 gRNA encoded on the
U6F1505 KM1F1
G1F2 505 KM1F1 557
KM2R1
Vg5’R2 Vg5’R1
KM2R1
557
G2R1 G2R2
kh2
m2A10 DsRed Cas9vasa m1C3 gRNAU6A
khW gene locus
wt 10.1 10.2 wt 10.1 10.2
wt 10.1 10.2
U6F1/KM2R1KM1F1/Vg5’R1 505/557
754 bp
sremirpsremirp
361bp
320 bp
wt 10.1 10.2 wt 10.1 10.2
G1F2/Vg5’R2 U6F1/G2R2
1599 bp
1170 bp
A
CB
HDR HDR
Fig. 1. Site-specific integration into the An. stephensi kynurenine hydroxy-
lase
white
locus of the gene-drive construct AsMCRkh2, carrying antimalarial
effector genes. (A) Schematic representations of the kynurenine hydroxy-
lase
white
locus and AsMCRkh2 construct. Genes and other features of the
AsMCRkh2 construct are not to scale. The dark red boxes represent the eight
exons of the endogenous kh
w
gene locus (Top) with the direction of tran-
scription indicated by the wedge in exon 8. The black lines represent ge-
nomic and intron DNA. The green arrowhead represents the target site of
the gRNA, kh2. Labels and arrows indicate names, approximate positions,
and directions of oligonucleotide primers used in the study. kh
w
gene se-
quences corresponding to previously characterized mutations are indicated
as an orange rectangle (28) and square (29). The plasmid, AsMCRkh2 (Bot-
tom), carries promoter and coding sequences comprising vasa-Cas9 and the
U6A-kh2 gRNA genes (U6A gRNA) linked to the dual scFv antibody cassette
(m2A10-m1C3) conferring resistance to P. falciparum (11) and the dominant
eye marker gene (DsRed) inserted between regions of homology (dark red
boxes) from the An. stephensi kh
w
locus that directly abut the U6A-kh2
gRNA cut site. The black lines represent kh
w
intron sequences, and the gray
lines indicate pla smid DN A sequences. Following gRNA- directed cleavage
by the Cas9–kh2 gRNA nuclease complex at the kh2 target site (green ar-
rowhead), homology-directed repair (HDR) leads to precise insertion of the
AsMCRkh2 cargo (m2A10-m1C3, DsRed, vasa-Cas9, U6A gRNA) into the ge-
nomic kh
w
locus via HDR events somewhere within the regions of homology
(pink-shaded quadrilaterals). Plasmid sequences are not integrated. (B)
Gene amplification analysis confirms integration of the AsMCRkh2 cargo in
genomic DNA prepared from the two G
1
male transformants (10.1 and 10.2)
that were positive for the DsRed eye-marker phenotype. Both males carry
left and right junction fragments of the AsMCRkh2 cargo with the supplied
kh
w
regions of homology (KM1F1/Vg5′R1 and U6F1/KM2R1 primer combi-
nations, respectively). An amplicon corresponding to the wild-type kh
w
locus
(505/557 primer pairs) confirms that these mosquitoes were heterozygous in
some of their cells. Wild-type (wt) control DNA supports amplification only
of the wild-type kh
w
locus (505/557 primers). (C) Gene amplification anal-
ysis con firms s ite-specific integration of the AsMCRkh2 construct at the
kh
w
locus using primers located outside of the genomic sequence included in
the AsMCRkh2 cassette (the left integration junction fragment amplified
with primers G1F2/Vg5′R2, and the right junction fragment amplified with
primers U6F1/G2R2). Wild-type control DNA did not support amplification of
these hybrid fragments. Numbers refer to the length in nucleotides of the
amplified fragments. Amplicon primary structure was verified by DNA se-
quencing (SI Appendix, Fig. S1).
2of8
|
www.pnas.org/cgi/doi/10.1073/pnas.1521077112 Gantz et al.

plasmid. A total of 122 and 129 adult males and females, re-
spectively (37%), survived to the adult stage. Adults were assigned
to 22 male-founder and 9 female-founder pools and outcrossed to
wild-type adults of the opposite sex. Two males positive for DsRed
fluorescence (DsRed
+
), designated 10.1 and 10.2, were re-
covered following screening of 25,712 G
1
larvae.
Efficient Autonomous Gene Drive of the AsMCRkh2 Construct. A
notable difference in the inheritance patterns of the DsRed
marker gene was observed in the G
2
progeny of the 10.1 and 10.2
G
1
males following outcrosses to wild-type females (SI Appendix,
Table S1). Male 10.1 produced all DsRed
+
adult progeny (n=14),
whereas male 10.2 produced 44% DsRed
+
progeny (57 of 129).
Although the number of 10.1 G
2
progeny is too small for statistical
analysis, the data are consistent with drive of the DsRed dominant
marker gene. However, the line 10.2 DsRed
+
ratios were not sig-
nificantly different from those expected of random Mendelian
segregation (Χ
2
=1.744, df =1; P=0.1866).
Target-specific integration of the transgene into the kh
w
locus
was verified in each of the G
1
founder males by gene amplifi-
cation using oligonucleotide primers complementary to DNA
within the construct and outside the regions of homology in-
cluded in the construct (Fig. 1 and SI Appendix, Table S2). Se-
quencing of the ends of the diagnostic amplicons spanning both
sides of the insertion within the kh
w
-coding region confirmed the
structure and precise integrity of the junctions of the transgene
and genomic DNAs (SI Appendix, Fig. S1).
DsRed
+
G
2
males and females derived from 10.1 and 10.2 G
1
founders were outcrossed individually and in batch matings to
wild-type mosquitoes, and G
3
larval progeny were scored for
DsRed. Extreme non-Mendelian DsRed segregation patterns were
evidentinboth10.1and10.2G
2
male and female outcrosses (SI
Appendix, Tables S3 and S4). Line 10.1 yielded 1,321 (99.7%)
DsRed
+
and 7 DsRed
−
G
3
larvae, whereas 10.2 produced 4,631
(99.2%) DsRed
+
and 35 DsRed
−
G
3
larvae. These highly biased
transmission frequencies deviate significantly from the 50% al-
lele inheritance expected of random segregation. Three DsRed
−
larvae with white eyes (kh
w−
) were recovered from female-
founder families, and gene amplification and sequencing con-
firmed that they have target site-specific deletions in the kh
w
locus consistent with Cas9-mediated NHEJ (SI Appendix,
Fig. S2).
Non-Mendelian segregation patterns consistent with gene
drive were also seen in the eye-color phenotypes of lines 10.1 and
10.2 G
3
larvae that survived to adults (Table 1 and SI Appendix,
Tables S5 and S6). Three major adult phenotypes consistent with
HDR were seen: mosquitoes positive for DsRed with an other-
wise wild-type eye color (DsRed
+
/kh
w+
), mosquitoes positive for
DsRed with a white eye color (DsRed
+
/kh
w−
), and mosquitoes
positive for DsRed with mosaicism evident in the eyes (DsRed
+
/
mosaic) (Fig. 2). Additionally, a number of G
3
progeny from fe-
male-founder families scored as DsRed
+
/kh
w−
phenotype (positive
for DsRed/white eye color) were modified by eye coloring (“col-
oring”;SI Appendix,TableS6). This coloring is consistent with
partial cell-nonautonomous rescue of the white-eye phenotype by
wild-type expression of the gene in a somatic location other than the
eye or a hypomorphic allele generated by NHEJ. The frequency of
this coloring phenotype in the 10 families in which it was explicitly
scoredvariedfrom5to32%,withanaverageof∼17%. The non-
drive phenotypic classes were mosquitoes negative for DsRed with a
wild-type eye color (DsRed
−
/kh
w+
) and the rare mosquitoes
negative for DsRed with a white eye color (DsRed
−
/kh
w−
) seen
in the larvae (Fig. 2 and Table 1).
The numerical summaries of the G
3
adult phenotypes confirm
the non-Mendelian segregation patterns consistent with highly
efficient gene drive. Male 10.1 and 10.2 DsRed
+
/kh
w+
single
founders and those batch-mated to wild-type females produced a
total of 2,113 G
3
progeny, 98.9% (2,091) of which were DsRed
+
(Table 1). None of the progeny derived from males had white or
mosaic eyes, indicating that these individuals were heterozygous
for the gene-drive construct. Individual DsRed
+
/kh
w+
10.1 and
10.2 female founders produced a total of 1,781 progeny, of which
1,778 (99.8%) were DsRed
+
. Notably, 5 of 7 of the 10.1 G
2
outcrosses and all 15 of the 10.2 female outcrosses had 100%
DsRed
+
G
3
progeny (SI Appendix, Tables S5 and S6). The
combined data from both male and female founders total 3,869
DsRed
+
G
3
progeny and 25 wild-type G
3
progeny, amounting to
∼99.5% efficiency of inheritance of the cargo facilitated by a 98.8%
DsRed+/khw- DsRed+/khw+
DsRed-/khw- DsRed+/mosaicsdeRsDepyt-dliW +/khw-
Wild-type
AC
DB
Fig. 2. Larval and adult phenotypes of AsMCRkh2 transgenic An. stephensi.
Bright-field and fluorescent images of larval (Aand B) and adult (Cand D)
eye-color phenotypes. All images are lateral views of the head. Phenotypic
descriptions are listed above. White arrows in the larval images indicate the
white-eye phenotype, and the yellow arrow indicates the wild-type eye
color. Note that all data presented in Tables 1 and 2 for the DsRed
+
phe-
notype are from scoring larvae, not adults. The white arrow in the adult
images indicates a patch of wild-type cells in a white-eye background of the
left mosaic. The right mosaic exemplifies the colored-eye phenotype.
Table 1. Summary of G
3
adult phenotypes of lines 10.1 and 10.2 G
2
outcrosses to wild-type mosquitoes
*Shaded cells are all G
3
progeny positive for DsRed (DsRed
+
).
†
Progeny of single G
2
founders outcrossed to wild-type counterparts.
‡
Twenty-seven G
2
males outcrossed to 270 wild-type females.
§
Χ
2
analyses show significant differences from random segregation.
Gantz et al. PNAS Early Edition
|
3of8
APPLIED BIOLOGICAL
SCIENCES
PNAS PLUS

gene-conversion rate. This latter value is derived from a formula:
conversion rate =2(X −0.5N)/N, where N is the total number of
mosquitoes and X is the number of DsRed-positive individuals,
which accounts for the fact that 50% of the progeny would be
expected to inherit the transgene by traditional Mendelian
segregation (26). These combined data support the conclusion
that Cas9-mediated gene conversion resulting from HDR can
occur at near-complete efficiency in the germ-line cells of both
male and female mosquitoes carrying the AsMCRkh2 cargo.
Remarkably, all but 3 of the 1,781 G
3
progeny from individual
DsRed
+
/kh
w+
10.1 and 10.2 female founders had either white or
mosaic eyes (DsRed
+
/kh
w−
phenotype; Table 1 and SI Appendix,
Tables S5 and S6). These data show that not only do the progeny
derived from phenotypically DsRed
+
/kh
w+
female founders
outcrossed to wild-type males transmit the gene-drive constructs
with high efficiency via the germ line but that the paternally
inherited kh
w+
gene is targeted somatically by the Cas9 nuclease
and gRNAs in the zygote and developing embryo, where it is
mutagenized by either NHEJ or HDR at a high efficiency (99.8%
of adult progeny). In contrast, no DsRed
+
/kh
w−
G
3
progeny were
recovered from the DsRed
+
/kh
w+
male-founder outcrosses. These
data support the interpretation that the eggs of transgenic
AsMCRkh2 females contain both Cas9 protein and kh2 gRNA.
Sex specificity (Cas9 presence in female but not male gametes)
of the somatic mutation phenotype is likely conferred by the vasa
regulatory sequences used for Cas9 expression, and this is con-
sistent with the expression profile of the orthologous vasa gene in
Anopheles gambiae (32).
Maternal Effects Result in Differential Transmission of the
pAsMCRkh2 Cargo. DsRed
+
/kh
w+
and DsRed
+
/kh
w−
10.1 and
10.2 G
3
males and females were outcrossed separately in batches
to wild-type mosquitoes of the appropriate opposite sex and G
4
progeny were scored both as larvae and adults. Larval screening
for the DsRed phenotype showed two distinct distributions of
the phenotypic classes depending on the history of the lineage.
G
4
larval progeny of G
3
males and females derived from G
2
10.1
and 10.2 transgenic males (crosses 5–8; SI Appendix, Tables S7
and S8) show a high frequency (98.5%) of DsRed transmission,
corresponding to a 96.9% rate of gene conversion. In contrast, a
much higher proportion of G
4
larval progeny of G
3
males and
females derived from G
2
10.1 and 10.2 transgenic females
(crosses 1–4; SI Appendix, Tables S7 and S8) appear to have
inherited mutations at the kh
w
locus instead of gene-conversion
events, as evidenced by inheritance ratios of 1.33:1 (936 DsRed
+
:
703 DsRed
−
) for the transgene cargo. However, this ratio still
deviates from that expected by Mendelian segregation alone
(X
2
=33.123, df =1; P<0.0001). We interpret these results to
indicate that the progeny of pAsMCRkh2 females often inherit
“indel”mutations presumably generated via the NHEJ pathway in
the male-derived kh
w
allele. However, the excess of DsRed
+
larvae
among the progeny is consistent with a fraction of the incoming
chromosomes also having been converted by HDR.
The presumed high level of NHEJ in G
4
progeny of DsRed
+
/
kh
w−
G
3
males and females derived from G
2
10.1 and 10.2
transgenic females (crosses 1–4; SI Appendix, Tables S7 and S8)
supports the hypothesis that the G
3
parents were at least partially
heterozygous for the DsRed cargo component of the transgene
and a nonconverted mutant kh
w
allele. Genomic DNA prepared
from 20 individual male and female DsRed
+
/kh
w−
G
3
founder
mosquitoes was used with gene-specific primers to amplify the
kh2 target portion of the kh
w
gene. Diagnostic fragments of 754
bp were seen in each of the samples, indicating that these mos-
quitoes had chromosomes without the large ∼17-kb transgene
cargo inserted into it (SI Appendix, Fig. S3). These fragments
must include mutant alleles, because the eye phenotype of each
mosquito from which the DNA was derived was white (kh
w−
).
The G
4
progeny of separate batch intercross matings of 10.1
and 10.2 DsRed
+
G
3
siblings yielded a combined total of 2,279
DsRed
+
and 432 DsRed
−
larvae (SI Appendix, Table S9). The
inheritance ratio, ∼5.3:1 (DsRed
+
:DsRed
−
), differs significantly
from 3:1 (Χ
2
=118.811, df =1; P<0.0001), that expected of an
intercross of two parents heterozygous for DsRed
+
. These data
provide further support for the conclusion that some level of
HDR continues to occur in the G
3
females derived from G
2
10.1
and 10.2 transgenic females.
The numerical summaries of the G
4
adult phenotypes confirm
the strong Cas9-mediated gene drive through both male and
female germ lines in individuals derived from wild-type males.
Male G
3
10.1 and 10.2 DsRed
+
/kh
w+
batch-mated to wild-type
females produced a total of 1,471 G
4
progeny, 98.4% (1,447) of
which were DsRed
+
(Table 2; crosses 6 and 8). As in previous
outcrosses of DsRed
+
cargo-bearing males, none of the progeny
had white or mosaic eyes, indicating that these individuals were
heterozygous for the gene-drive construct in somatic tissues.
Female G
3
10.1 and 10.2 DsRed
+
/kh
w+
batch-mated to wild-type
males produced 1,523 adult G
4
progeny, 98.8.% (1,505) of which
were DsRed
+
(Table 2; crosses 5 and 7). Additionally, 1,500
(99.7%) of the DsRed
+
mosquitoes derived from DsRed
+
/kh
w+
mothers had white or mosaic/colored eyes, a result consistent
with previous outcrosses of this type. The combined data from
both DsRed
+
/kh
w+
male and female G
3
outcrosses total 2,952
DsRed
+
and 42 wild-type G
4
progeny, amounting to ∼98.6%
efficiency of inheritance of the cargo and corresponding to a
97.2% rate of gene conversion. These data provide strong sup-
port for the conclusion that Cas9-mediated gene drive continues
Table 2. Summary of G
4
adult phenotypes of lines 10.1 and 10.2 G
3
outcrosses to wild-type mosquitoes
*Shaded cells are all G
4
progeny positive for DsRed (DsRed
+
).
†
Crosses are listed in Fig. 3 and SI Appendix, Tables S10 and S11.
4of8
|
www.pnas.org/cgi/doi/10.1073/pnas.1521077112 Gantz et al.

to occur efficiently and in a multigenerational fashion in the germ
line of these transgenic mosquitoes.
The reduced germ-line transmission of the AsMCRkh2 cargo
in G
4
larvae derived from DsRed
+
/kh
w−
G
3
parents described
above also was evident in the adult phenotypes. The ratio (1.27:1) of
DsRed
+
/DsRed
−
phenotypes of the corresponding adult G
4
prog-
eny derived from DsRed
+
/kh
w−
males (Table 2; crosses 2 and 4; SI
Appendix, Tables S10 and S11) still deviates from that expected by
Mendelian segregation alone (Χ
2
=16.404, df =1; P<0.0001).
Similarly, G
4
adult progeny of DsRed
+
/kh
w−
females (Table 2;
crosses 1 and 3; SI Appendix,TablesS10andS11) had a DsRed
+
/DsRed
−
phenotypic ratio of 1.48:1, also significantly different from
that expected solely by Mendelian segregation (Χ
2
=11.014, df =
1; P=0.0009). However, in contrast to the crosses with male G
3
parents in which all DsRed
−
progeny were kh
w+
, 41.2% (49/119)
of the DsRed
−
progeny of G
3
females had white eyes (kh
w−
).
These data provide further support for the conclusion that Cas9–
gRNA complexes perdure in eggs derived from transgenic cargo-
bearing females. The relatively fewer number of progeny derived
from the DsRed
+
/kh
w−
females may be indicative of a load asso-
ciated with this genotype.
The differences and consequences of the maternal effects on
gene drive are summarized in Fig. 3. In all cases where the
AsMCRkh2 cargo is propagated by outcrossing DsRed
+
/kh
w+
males to wild-type females, high-frequency DsRed
+
/kh
w+
prog-
eny are recovered. This inheritance of the cargo is consistent
with HDR gene drive and extends through an additional gen-
eration. In contrast, propagation of the AsMCRkh2 cargo by
outcrossing DsRed
+
/kh
w+
females to wild-type males produces a
high frequency of DsRed
+
/kh
w−
along with somatic mosaicism.
Continued outcrossing of these individuals to wild-type mos-
quitoes results in progeny inheriting the AsMCRkh2 cargo in
ratios approaching Mendelian segregation. Although some degree
of gene drive is observed in these mosquitoes, HDR-mediated
copying of the AsMCRkh2 cargo is reduced (typically ∼12–25%
conversion assayed in larvae and adults; Table 2) relative to crosses
in which that construct has been propagated with high fidelity via
DsRed
+
/kh
w+
parents. The most likely explanation for this differ-
ence is that in crosses where the AsMCRkh2-bearing parent is fe-
male, Cas9 protein is presumably expressed throughout the
cytoplasm of the egg, where it may generate indel mutations via
NHEJ that disrupt the gRNA cleavage site and thereby preclude
subsequent HDR-mediated copying of the cargo in the germ-
line lineages.
The Antipathogen Effector Genes Are Transcriptionally Active. The
m1C3 and 2A10 scFvs are under the control of the blood meal-
inducible 5′- and 3′-end regulatory elements of the An. gambiae
carboxypeptidase A (AgCPA) and An. stephensi Vitellogenin 1
(AsVg1) genes, respectively (11). Blood meal-induced, tissue-
specific accumulations of m1C3 and 2A10 transcripts were ob-
served by RT-PCR analysis of total RNA isolated from G
3
DsRed
+
/kh
w−
dissected females and whole males (Fig. 4). Mid-
gut and carcass (all tissues except the midguts) were collected
from females at 0, 4, 12, 24, and 48 h post blood meal (hPBM).
The AgCPA promoter driving expression of the m1C3 scFv dis-
plays constitutive midgut-specific expression in the absence of a
blood meal but increases to a peak at 4 hPBM and falls over the
next 2 d, consistent with the endogenous expression profile of
the orthologous gene from An. stephensi (10). Expression of the
2A10 transgene product by the AsVg1 promoter shows induction
at 12 hPBM with a qualitative maximum at 24 hPBM in carcasses
and an earlier induction in the midgut. Because midgut expres-
sion is not a characteristic feature of the endogenous gene (33),
position effects resulting from the insertion site may contribute
to this result. As expected, samples from males show no ex-
pression from either transgene. These data support the conclu-
sion that the antipathogen effector genes are transcribed in a
blood meal-regulated fashion following Cas9-mediated integration.
Fig. 3. Phenotypic inheritance patterns of the AsMCRkh2 gene-drive cargo.
(Top) DsRed-positive G
2
transgenic adult males and females with wild-type
eye color (DsRed
+
/kh
w+
, half-red and half-black circles) were outcrossed to
wild-type mosquitoes (all-black circles) of the opposite sex. (Middle,Upper)
G
3
progeny resulting from the male outcrosses were predominantly
DsRed
+
/kh
w+
(half-red and half-black circles; Table 1). G
3
progeny resulting from
the female outcrosses were predominantly positive for DsRed and had white
(DsRed
+
/kh
w−
, half-red and half-white circles) or mosaic eyes (DsRed
+
/mosaic,
half-red and half-checkered white and black circles). (Middle, Lower)DsRed
+
/kh
w+
(half-red and half-black circles) and DsRed
+
/kh
w−
(half-red and half-white cir-
cles) G
3
adult males and females were outcrossed to wild-type mosquitoes (all-
black circles) of the opposite sex. Specific crosses and tables for the data are
referenced. (Bottom)G
4
progeny resulting from outcrosses of DsRed
+
/kh
w+
G
3
adult males (half-red and half-black circles; crosses 6 and 8) were pre-
dominantly DsRed
+
/kh
w+
(half-red and half-black circles), whereas those from
G
3
DsRed
+
/kh
w+
adult females (half-red and half-black circles; crosses 5 and 7)
were predominantly positive for DsRed and had white (DsRed
+
/kh
w−
, half-red
andhalf-whitecircles)ormosaiceyes(DsRed
+
/mosaic, half-red and half-
checkered white and black circles). In contrast, G
4
progeny resulting from
outcrosses of DsRed
+
/kh
w−
G
3
adult males (crosses 2 and 4) were either
DsRed
+
/kh
w+
(half-red and half-black circles) or DsRed
−
/kh
w+
(wild-type eye,
all-black circles). G
4
progeny derived from female DsRed
+
/kh
w−
outcrosses
(crosses 1 and 3) were also a mix of DsRed
+
and DsRed
−
. Nearly all DsRed
+
progeny had white (DsRed
+
/kh
w−
, half-red and half-white circles) or mosaic
(DsRed
+
/mosaic, half-red and half checkered circles) eyes (Table 1). Among the
DsRed
−
progeny, approximately half had wild-type eyes (all-black circles) and
half had white eyes (half-black and half-white circles). Male-derived (G
2
cross)
G4 progeny (Left) show a bias of HDR over NHEJ, whereas female-derived (G
2
cross) G
4
progeny lines display nearly equal HDR and NHEJ.
AsVg1-m2A10
AgCPA-m1C3
S26
ssa
craCtu
gdi
M
Male 0 4 12 24 48 0 4 12 24 48
Fig. 4. Expression of m1C3 and m2A10 transcripts in AsMCRkh2 transgenic
females. RT-PCR was used to detect m1C3 (AgCPA-m1C3) and m2A10
(AsVg1-m2A10) transcripts in RNA isolated from homogenates of dissected
midguts and the remaining carcasses of mixed heterozygous and homozy-
gous DsRed
+
G
3
females at 0 (non–blood-fed), 4, 12, 24, and 48 h post blood
meal. Male transgenic mosquitoes were used as negative controls. The
An. stephensi S26 ribosomal protein transcript was amplified from all sam-
ples as a loading control.
Gantz et al. PNAS Early Edition
|
5of8
APPLIED BIOLOGICAL
SCIENCES
PNAS PLUS

Discussion
The data presented here support the following conclusions: (i)Cas9-
mediated gene drive based on a system adapted from MCR works
well in a malaria mosquito, An. stephensi;(ii) the gene-drive sys-
tem is target-specific; (iii) the system works in the germ line of
both males and females; (iv) the system is active early in the so-
matic cells of embryos derived from transgenic females; (v)the
gene-drive system can carry a relatively large cargo; and (vi)the
cargo is functional (at the transcriptional level). These results
provide the basis for the further development of Cas9-mediated
gene-drive technology in sustaining malaria control and elimina-
tion as part of the eradication agenda.
The use of both dominant and recessive marker genes for gene-
drive function, as well as the choice of promoter for driving the Cas9
activity, has provided a number of insights that can guide the further
development of this approach into a functional system for malaria
control. The tight linkage of the DsRed marker gene to the anti-
pathogen effector scFvs allows the tracking of the malaria-resistance
phenotype by monitoring fluorescence in samples of larvae. This is
expected to have significant practical value as this technology con-
tinues to be developed for the field. Targeting the kh
w
gene allowed
us to monitor the specificity of Cas9-mediated gene conversion and
mutagenesis by scoring the white-eye phenotype. Furthermore, the
data presented here provide support for the hypothesis that there is
a load associated with the white-eye phenotype (homozygous kh
w−
),
so this locus may not be optimal for any strain developed for field
applications. Other target sites are available in the An. stephensi
genome that appear not to have any significant fitness issues (10,
11, 21), and further validation of the technology may make it
unnecessary to target loci with recessive visible phenotypes.
The sex-specific expression of Cas9 mediated by the An. ste-
phensi vasa ortholog control DNA sequences was highly in-
formative for the design of future autonomous gene-drive systems.
Previous transgene analysis of the An. gambiae vasa ortholog
showed that it is active in both male and female germ-line tis-
sues, and that sex- and tissue-specific enhancer-like sequences
could be localized in the promoter and 5′untranslated regions
(5′UTRs) (32). We chose to use the complete promoter and
5′UTR sequences of the An. stephensi ortholog in our autono-
mous construct to maximize the potential for germ-line gene con-
version via HDR. This allowed us to discover the effects of pre- and
postzygotic activity of the Cas9 nuclease and gRNAs and the impact
of HDR and NHEJ on subsequent gene drive and inheritance.
Our data support a model in which the gene drive and in-
heritance of the transgene cargo are optimized in the progeny of
transgenic males whose female parent was wild-type (Fig. 5).
Male and female progeny of these males also faithfully trans-
mitted the cargo to their progeny. However, progeny of trans-
genic males and females whose parent was a transgenic female
transmit the cargo in ratios similar to what is expected of Men-
delian segregation, although there does appear to be some re-
sidual drive. These differences can be understood in terms of a
maternal effect of Cas9 expression in the developing embryos.
Outcrosses of transgenic male parents to wild-type females result
in an embryonic environment in which the eggs lack maternally
produced Cas9. Hence, Cas9 expression is restricted to the germ
line, where it continues to catalyze high-frequency HDR. In
contrast, outcrosses of transgenic female parents to wild-type
males produce embryos in which the eggs have active levels of
Cas9 and kh2 gRNAs. The wild-type kh
w
(kh
w+
) allele contrib-
uted by the sperm can be subject to Cas9-mediated activity in
pre- and postsyncytial blastoderm nuclei before cellularization
and partitioning of the germplasm into posterior cells to form the
germ line. This early activity could result in either HDR or NHEJ.
Given the initial physical separation of the paternally derived kh
w+
allele from the maternally derived allele immediately following
fertilization, the repair template (the DsRed
+
,kh
w−
allele) may be
positioned sufficiently far from the Cas9-induced double-stranded
break and favor NHEJ. The fact that nearly all progeny from such
crosses manifest a white-eye phenotype supports the interpreta-
tion that such early-acting mutagenesis occurs at a high frequency.
Importantly, once a homologous chromosome has been mutated
by NHEJ, key nucleotides required for gRNA recognition will
typically be eliminated, thus precluding subsequent HDR-mediated
gene conversion in the germ line. This has a dampening effect on
drive, and progeny phenotype ratios thus approach Mendelian in-
heritance. This also explains the rare phenotypes we observed in the
female-derived lines.
Fig. 5. Model of AsMCRkh2 transgene activity in adult males and females.
(Top) Schematic representations of the third chromosomes of An. stephensi.
Transgenic males (Left)andfemales(Right) are depicted as being homozygous
in the germline for AsMCRkh2 (red bars) and are outcrossed to wild-type mos-
quitoes of the opposite sex. Zygotes resulting from outcrosses of transgenic
males do not have the Cas9 nuclease in the eggs (clear oval), which are derived
from wild-type females, and somatic cells remain heterozygous for the
AsMCRkh2 transgene. A schematic representation of the sperm attached
to the egg and the donated paternal chromosome is represented encircled by
the dotted line. vasa-mediated expression of Cas9 is restricted to the germ line
(colored half-oval) in developing embryos derived from transgenic AsMCRkh2
males, resulting in significant HDR (red arrowhead) that converts the majority of
the chromosomes by insertion of the AsMCRkh2 cargo. Adults are phenotypi-
cally positive for the dominant reporter gene, DsRed, and wild-type in eye color.
In contrast, zygotes resulting from outcrosses of transgenic females have Cas9
nuclease in the eggs (aqua-colored oval) as a result of vasa-directed expression
in the maternal germ line, and this catalyzes nonhomologous end joining (as-
terisk) to mutate the paternally derived wild-type chromosome (encircled by the
dotted line). Some HDR may occur at this stage, but may be hampered by an
initial physical separation of the maternal and paternal chromosomes. Embryos
derived from transgenic AsMCRkh2 females also have vasa-mediated Cas9 ex-
pression restricted to the germ line (colored half-oval), but in addition have the
nuclease perduring from the maternal gamete (light-colored half-oval), which
can result in adults that are phenotypically positive for the dominant reporter
gene, DsRed, and exhibit the white or mosaic eye color. Furthermore, the pa-
ternally derived chromosomes mutagenized in the zygotes are resistant to
subsequent HDR and insertion of the cargo. Some of the male-derived chro-
mosomes may not be mutagenized, and these can be substrates for HDR. Both
options are shown as the asterisk overlying the red bar in the germ line.
6of8
|
www.pnas.org/cgi/doi/10.1073/pnas.1521077112 Gantz et al.

Two types of mosaic phenotypes result in those animals where
mutagenesis of the kh
w
locus is not complete. First, patches of
wild-type cells visible in the eyes reflect groups of cells in the eye
in which there was no mutagenesis. Second, uniform colored-eyed
phenotypes may arise as a consequence of the kynurenine hy-
droxylase enzyme being diffusible throughout the insect (28), in
which case the colored eyes could arise from patches of wild-type
cells outside the eye contributing enough enzyme to produce
some pigment in the eyes. Alternatively, NHEJ may generate
partial loss-of-function alleles whose products synthesize reduced
levels of pigment.
An observation that does not have a straightforward expla-
nation is the difference in inheritance seen in the G
2
progeny of
the original 10.1 and 10.2 G
1
founder males. One male founder
(10.1) transmitted the construct to all of its G
2
progeny, whereas
the other (10.2) transmitted the element in a Mendelian fashion.
The insertion events of both of these males are precise based on
sequencing of junction fragments in genomic DNA. Further-
more, the G
2
progeny of these males efficiently propagated the
cargo to G
3
male and female progeny, indicating that both in-
sertional events had functional converting elements. Because
DsRed
+
G
2
10.2 males and females transmitted the cargo at the
same high frequency as their G
2
10.1 DsRed
+
counterparts, the
differences in transmission observed between the two G
1
foun-
ders did not result in heritable differences in the transgene or its
insertion site. It is possible that short-duration epigenetic dif-
ferences between the two insertional events or differential per-
sistence of injected Cas9 dsRNA may account for the observed
transgene inheritance. The recovery of DsRed
−
/kh
w−
G
4
progeny
from DsRed
+
/kh
w−
females is consistent with transgenerational
perdurance of Cas9–gRNA complexes.
A number of alternate approaches could mitigate the maternal
effects that result in a high frequency of NHEJ and drive-resistant
loci. The most straightforward solution is to drive Cas9 with a male
germ-line–specific cis-regulatory sequence such as those of the well-
characterized β2-tubulin gene, which is expressed only in the sperm
of dipterans including D. melanogaster (34–36), An. stephensi (37),
and Ae. aegypti (38). Alternatively, it may be possible to reduce the
activity of Cas9 in the egg by maternal (but not germ-line) ex-
pression of Cas9 and/or Ku70 RNAi constructs. This is similar to
the approach applied here for the initial recovery of the AsMCRkh2
transgenic mosquitoes. Advances in CRISPR/Cas9 research also
offer potential solutions that greatly reduce the inhibition of
HDR by the prior action of NHEJ by exploiting modified nu-
cleases that cleave DNA at a distance from the gRNA recog-
nition sequence, thereby allowing multiple rounds of target
mutagenesis without eliminating the gRNA target site (39), or
those that are mutated to cleave a single strand, the so-called
nicking endonucleases (40). Autonomous gene-drive constructs
using these modified nucleases may be less susceptible to NHEJ
alteration of the homologous chromosome, which could then re-
main an efficient target for subsequent HDR-mediated conversion
in the germ line. Future tests of these various strategies should
establish those approaches that are the most effective for robust
multigenerational maintenance of gene drive.
Gene-drive systems for population modification of vector
mosquitoes have been proposed for nearly half a century (41).
The phenomenal rate of allelic conversion achieved is a mile-
stone achievement in the development of population-modifica-
tion strategies for controlling malaria and other vector-borne
diseases. These efforts justify a degree of optimism for the future
successful application of this technology. We are fully aware that
much needs to be done before laboratory achievements of this
type are moved to the field. Effector gene stability in different
genetic backgrounds and under diverse environmental conditions
and efficacy against genetically diverse parasites need further
research to ensure that the constructs function as well in the field
as they do in the laboratory. In addition, significant advances in
regulatory structures and ethical models of community engage-
ment are as important as the further scientific development of
these technologies (7, 42, 43). It is incumbent on the scientist de-
veloping these technologies to interact openly and freely with the
potential end users. Finally, we do not believe that these tech-
nologies alone will be sufficient for malaria eradication. We support
the combined efforts of people developing prophylactic and thera-
peutic drugs, vaccines, and alternate vector-control measures.
Materials and Methods
Mosquito Rearing and Maintenance. A colony of An. stephensi (15) bred in our
insectary for >7 y was used in the experiments. The mosquitoes were
maintained at 27 °C with 77% humidity and a 12-h day/night, 30-min dusk/
dawn lighting cycle. Larvae were fed a diet of powdered fish food (Tetra-
Min) mixed with yeast. Adults were provided with water and a 10% (wt/vol)
sucrose solution ad libitum. Blood meals were provided by artificial feeding
or mice. Protocols were approved by the Intuitional Animal Care and Use
Committee of the University of California, Irvine (NIH Animal Welfare
Assurance no. A3416.01). Mosquito containment followed recommended
procedures (44).
Oligonucleotide Primers. SI Appendix, Table S2 lists oligonucleotide primer
names and sequences used for gene amplification.
Design of the Gene-Drive Construct. Standard molecular biological procedures
were used to construct the gene-drive plasmid, pAsMCRkh2. Detailed de-
scriptions of the construction and plasmid architecture are provided in SI
Appendix, Materials and Methods.
Generation of Double-Stranded RNA. Double-stranded RNA was generated to
inhibit the expression of Cas9 from the donor plasmid during microinjection.
Previous injections using Cas9 expressed from a plasmid or from Cas9 mRNA
were unsuccessful, and we hypothesized that a high amount of Cas9 could be
toxic to the embryos. Therefore, a total of 100 ng/μL Cas9 protein was in-
cluded in the injection mixture along with dsRNA targeting Cas9 mRNA so
that Cas9 mRNA generated by expression from the donor plasmid in the
embryo would be destroyed and only the injected protein would be present.
Additionally, dsRNA was injected targeting the putative ortholog of Ku70, a
protein essential for nonhomologous end joining (45, 46). Knockdown of
this protein may increase the possibility of repair by homologous recombi-
nation (47, 48). The putative ortholog for Ku70 in An. stephensi, ASTE011109,
was identified by homology to Bombyx mori X-ray repair cross-complementing
protein 5-like mRNA (LOC101736121) using the BLAST tool available at
VectorBase.org. Primers were designed to amplify a 561-bp region of the
ASTE011109 transcript and a 637-bp region of the An. stephensi codon-
optimized Cas9, such that the amplicons did not contain >19 bp of identity
to any other putative transcript available on VectorBase to avoid off-target
effects. Both forward and reverse primers have T7 promoters at the 5′end.
The amplification products were purified using DNA Clean & Concentrator
(Zymo) and used as a template for reverse transcription using the RNAi Kit
from Ambion.
Microinjection and Screening Procedures. Microinjections were performed as
described previously (10). Embryos were injected with a solution containing
100 ng/μL each of the plasmid, pAsMCRkh2, Cas9 protein, Cas9 dsRNA, and
Ku70 dsRNA. G
0
males and females were outcrossed to wild-type mosquitoes
in pools of ∼5G
0
males or 15–30 G
0
females. All G
1
,G
2
,G
3
, and G
4
progeny
were screened as larvae for DsRed fluorescence under UV-fluorescence mi-
croscopy, and adults were screened under light microscopy.
RT-PCR. RT-PCR analyses were adapted from those used in ref. 11 with an
additional RNA purification with Zymo RNA Clean & Concentrator. A total of
15 males and 30 female carcasses and dissected midguts from mixed het-
erozygous and homozygous DsRed
+
G
3
mosquitoes was used for each RNA
preparation. Two hundred nanograms of DNase-treated total RNA was used
in each reaction.
ACKNOWLEDGMENTS. The authors are grateful to Judy Coleman and Thai
Binh Pham for mosquito husbandry. Research was supported by grants
from the NIH (AI070654 and NS029870), a generous gift from Drs. Sarah
Sandell and Michael Marshall (to E.B.), the W. M. Keck Foundation, an d t he NIH
National Institute of Allergy and Infectious Diseases (AI29746 and AI116433
to A.A.J.).
Gantz et al. PNAS Early Edition
|
7of8
APPLIED BIOLOGICAL
SCIENCES
PNAS PLUS

1. White NJ, et al. (2014) Malaria. Lancet 383(9918):723–735.
2. World Health Organization (2014) World Malaria Report, 2013 (WHO, Geneva,
Switzerland).
3. World Health Organization (2015) World Malaria Report, 2014 (WHO, Geneva,
Switzerland).
4. Nájera JA (2001) Malaria control: Achievements, problems and strategies. Parassitologia
43(1-2):1–89.
5. malERA Consultative Group on Vector Control (2011) A research agenda for malaria
eradication: Vector control. PLoS Med 8(1):e1000401.
6. Luckhart S, Lindsay SW, James AA, Scott TW (2010) Reframing critical needs in vector
biology and management of vector-borne disease. PLoS Negl Trop Dis 4(2):e566.
7. Macias VM, James AA (2015) Impact of genetic modification of vector populations
on the malaria eradication agenda. Genetic Control of Malaria and Dengue,ed
Adelman ZN (Elsevier Academic, San Diego), pp 423–444.
8. Collins FH, James AA (1996) Genetic modification of mosquitoes. Sci Med 3(6):52–61.
9. James AA, et al. (1999) Controlling malaria transmission with genetically-engineered,
Plasmodi um-resistant mosquitoes: Milestones in a model sy stem. Parassitologia
41(1-3):461–471.
10. Isaacs AT, et al. (2011) Engineered resistance to Plasmodium falciparum development
in transgenic Anopheles stephensi.PLoS Pathog 7(4):e1002017.
11. Isaacs AT, et al. (2012) Transgenic Anopheles stephensi coexpressing single-chain
antibodies resist Plasmodium falciparum development. Proc Natl Acad Sci USA
109(28):E1922–E1930.
12. Ito J, Ghosh A, Moreira LA, Wimmer EA, Jacobs-Lorena M (2002) Transgenic anoph-
eline mosquitoes impaired in transmission of a malaria parasite. Nature 417(6887):
452–455.
13. Corby-Harris V, et al. (2010) Activation of Akt signaling reduces the prevalence and
intensity of malaria parasite infection and lifespan in Anopheles stephensi mosqui-
toes. PLoS Pathog 6(7):e1001003.
14. James AA, Peloquin JJ (1998) Engineering resistance to malaria parasite development
in mosquitoes. Malaria: Parasite Biology, Pathogenesis and Protection, ed Sherwin IW
(ASM, Washington, DC), pp 63–69.
15. Jiang X, et al. (2014) Genome analysis of a major urban malaria vector mosquito,
Anopheles stephensi.Genome Biol 15(9):459.
16. Sharma VP (1999) Current scenario of malaria in India. Parassitologia 41(1-3):349–353.
17. Gakhar SK, Sharma R, Sharma A (2013) Population genetic structure of malaria vector
Anopheles stephensi Liston (Diptera: Culicidae). Indian J Exp Biol 51(4):273–279.
18. Murray CJ, et al. (2012) Global malaria mortality between 1980 and 2010: A system-
atic analysis. Lancet 379(9814):413–431.
19. Faulde MK, Rueda LM, Khaireh BA (2014) First record of the Asian malaria vector
Anopheles stephensi and its possible role in the resurgence of malaria in Djibouti,
Horn of Africa. Acta Trop 139:39–43.
20. Catteruccia F, et al. (2000) Stable germline transformation of the malaria mosquito
Anopheles stephensi.Nature 405(6789):959–962.
21. Amenya DA, et al. (2010) Comparative fitness assessment of Anopheles stephensi
transgenic lines receptive to site-specific integration. Insect Mol Biol 19(2):263–269.
22. Hollingdale MR, Nardin EH, Tharavanij S, Schwartz AL, Nussenzweig RS (1984) In-
hibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured
cells; an in vitro assay of protective antibodies. J Immunol 132(2):909–913.
23. Li F, Patra KP, Vinetz JM (2005) An anti-Chitinase malaria transmission-blocking sin-
gle-chain antibody as an effector molecule for creating a Plasmodium falciparum-
refractory mosquito. J Infect Dis 192(5):878–887.
24. Robert MA, Okamoto KW, Gould F, Lloyd AL (2014) Antipathogen genes and the
replacement of disease-vectoring mosquito populations: A model-based evaluation.
Evol Appl 7(10):1238–1251.
25. Klassen W, Curtis CF (2005) History of the sterile insect technique. Sterile Insect
Technique: Principles and Practice in Area-Wide Integrated Pest Management, eds
Dyck VA, Hendrichs J, Robinson AS (Springer Dordrecht, The Netherlands), pp 3–36.
26. Gantz VM, Bier E (2015) Genome editing. The mutagenic chain reaction: A method for
converting heterozygous to homozygous mutations. Science 348(6233):442–444.
27. Bhalla SC (1968) White eye, a new sex-linked mutant of Aedes aegypti.Mosq News
28(3):380–385.
28. Han Q, et al. (2003) Analysis of the wild-type and mutant genes encoding the enzyme
kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti.Insect Mol
Biol 12(5):483–490.
29. Aryan A, Anderson MA, Myles KM, Adelman ZN (2013) TALEN-based gene disruption
in the dengue vector Aedes aegypti.PLoS One 8(3):e60082.
30. Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes
Evol 210(12):630–637.
31. Basu S, et al. (2015) Silencing of end-joining repair for efficient site-specific gene
insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.Proc Natl Acad Sci USA
112(13):4038–4043.
32. Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A (2009) The vasa
regulatory region mediates germline expression and maternal transmission of pro-
teins in the malaria mosquito Anopheles gambiae: A versatile tool for genetic control
strategies. BMC Mol Biol 10:65.
33. Nirmala X, et al. (2006) Functional characterization of the promoter of the vitello-
genin gene, AsVg1, of the malaria vector, Anopheles stephensi.Insect Biochem Mol
Biol 36(9):694–700.
34. Kemphues KJ, Kaufman TC, Raff RA, Raff EC (1982) The testis-specific beta-tubulin
subunit in Drosophila melanogaster has multiple functions in spermatogenesis. Cell
31(3 Pt 2):655–670.
35. Michiels F, Gasch A, Kaltschmidt B, Renkawitz-Pohl R (1989) A 14 bp promoter ele-
ment directs the testis specificity of the Drosophila beta 2 tubulin gene. EMBO J 8(5):
1559–1565.
36. Santel A, Kaufmann J, Hyland R, Renkawitz-Pohl R (2000) The initiator element of the
Drosophila beta2 tubulin gene core promoter contributes to gene expression in vivo
but is not required for male germ-cell specific expression. Nucleic Acids Res 28(6):
1439–1446.
37. Catteruccia F, Benton JP, Crisanti A (2005) An Anopheles transgenic sexing strain for
vector control. Nat Biotechnol 23(11):1414–1417.
38. Smith RC, Walter MF, Hice RH, O’Brochta DA, Atkinson PW (2007) Testis-specific ex-
pression of the beta2 tubulin promoter of Aedes aegypti and its application as a
genetic sex-separation marker. Insect Mol Biol 16(1):61–71.
39. Zetsche B, et al. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-
Cas system. Cell 163(3):759–771.
40. Cong L, et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science
339(6121):819–823.
41. Curtis CF (1968) Possible use of translocations to fix desirable genes in insect pest
populations. Nature 218(5139):368–369.
42. Benedict M, et al. (2014) Guidance Framework for Testing of Genetically Modified
Mosquitoes (WHO/TDR, Geneva, Switzerland).
43. Brown DM, Alphey LS, McKemey A, Beech C, James AA (2014) Criteria for identifying
and evaluating candidate sites for open-field trials of genetically engineered mos-
quitoes. Vector Borne Zoonotic Dis 14(4):291–299.
44. Akbari OS, et al. (2015) Safeguarding gene drive experiments in the laboratory.
Science 349(6251):927–929.
45. Lieber MR (2010) The mechanism of double-strand DNA break repair by the non-
homologous DNA end-joining pathway. Annu Rev Biochem 79:181–211.
46. Williams GJ, et al. (2014) Structural insights into NHEJ: Building up an integrated
picture of the dynamic DSB repair super complex, one component and interaction at a
time. DNA Repair (Amst) 17:110–120.
47. Adelman ZN, et al. (2007) nanos gene control DNA mediates developmentally regu-
lated transposition in the yellow fever mosquito Aedes aegypti.Proc Natl Acad Sci
USA 104(24):9970–9975.
48. Ma S, et al. (2014) CRISPR/Cas9 mediated multiplex genome editing and heritable
mutagenesis of BmKu70 in Bombyx mori.Sci Rep 4:4489.
8of8
|
www.pnas.org/cgi/doi/10.1073/pnas.1521077112 Gantz et al.

ϭ

6XSSRUWLQJ,QIRUPDWLRQ$SSHQGL[
7DEOH6/DUYDODQGDGXOWSKHQRW\SHVRI*SURJHQ\RIOLQHVDQG*RXWFURVVHVWR
ZLOGW\SHPRVTXLWRHV
7DEOH6/LVWRIROLJRQXFOHRWLGHSULPHUV
7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*ODUYDOSKHQRW\SHVRIOLQHVDQGEDWFK*LQWHUFURVVHV
7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
)LJXUH60ROHFXODUFRQILUPDWLRQRIWKHSUHFLVHLQVHUWLRQRIWKH$V0&5NKFDUJRLQWRWKH
khwORFXV
)LJXUH61XFOHRWLGHVHTXHQFHVRIWKHNKWDUJHWVLWHVLQVHOHFWHG*JHQHGULYH
SKHQRW\SHV
)LJXUH6*HQHDPSOLILFDWLRQRIWKHNKWDUJHWVLWHVLQVHOHFWHG*IRXQGHUPRVTXLWRHV
6XSSRUWLQJ0DWHULDOVDQG0HWKRGV
5HIHUHQFHV

Ϯ

7DEOH6/DUYDODQGDGXOWSKHQRW\SHVRI*SURJHQ\RIOLQHVDQG*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
*
)RXQGHU
/DUYDOSKHQRW\SHV$GXOWSKHQRW\SHV
0DOHV )HPDOHV
'V5HGNKZ'V5HGNK
Z
 'V5HGNKZ'V5HG
NK
Z
'V5HGNKZ'V5HGNK
Z
'V5HG
NKZ'V5HG
NK
Z
'V5HGNKZ'V5HGNK
Z
'V5HG


NKZ 'V5HGNK
Z

ƃ    
ƃ    
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG


ϯ

7DEOH6/LVWRIROLJRQXFOHRWLGHSULPHUV
3ULPHU6HTXHQFH¶¶5HIHUHQFH
9J *7$&*&*7$7&*$7$$*&77WDD*$7$&$77*$7*$*777** 7KLVVWXG\
9J 7$**&&**&&*$7&7&**$7&7*$&$$7* 7KLVVWXG\
9J *&77$7&*$7$&*&*7$&*&$7&7*&$7&&7**7$&&$&$*7&77$77** 7KLVVWXG\
9J *7$&77&77$7&&$7&77&&7$*$7&*$7777$$*&$&*&&*& 7KLVVWXG\
9J &7$**$$*$7**$7$$*$$*7$&7&*$7&**7&7**$7$7&** 7KLVVWXG\
9J &7&7&*77$&$&&77*&*&77&77&77&**&* 7KLVVWXG\
9J *&$$**7*7$$&*$*$*77&**7*&*$$7&7&7&7&77*$77777&&& 7KLVVWXG\
9J $*$7&**&&**&&7$$&&$$**&&$*&&7*77*$*& 7KLVVWXG\
9J $$*&77$7&*$7$&*&*7$&&7&$$&7*&&*$&*$*77*&7&* 7KLVVWXG\
9J $7&&*$*$7&**&&**&&7$&*77&7*&*7**&7*77*7$$**77&& 7KLVVWXG\
9J **7$7&$*&7&$&7&$$$**&**7$$7$&** 7KLVVWXG\
9J *$$$***&&7&*7*$7$&*&&7$77777$7$** 7KLVVWXG\
9J &&777*$*7*$*&7*$7$&&&&&$$$$$77*$&&7&$*$7&7&7&$$*$$*&* 7KLVVWXG\
9J *&*7$7&$&*$**&&&777&*&$7&7*$$&*$**7*&7*&7$$$7** 7KLVVWXG\
9J *7777$*$*&7$*$$$7$*&$$*77$$$$7$$**&7$*7&&*77$7&$$&77*$$$$$*7**&$&
&*$*7&**7*&7777777*7**$$$777*$77&$&77*7777$** 7KLVVWXG\
9J $**7&77&7&*$$*$&&&&$$***$*****&$$$7***77** 7KLVVWXG\
9J *7&&$&7$$&*$$$*$**7&$$*$*& 7KLVVWXG\
9J &*$7&*777$*7*$&*$*$7&$&*& 7KLVVWXG\
9J &77*$7**77&&*77&7$&***& 7KLVVWXG\
9J $$$&*&&&*7$*$$&**$$&&$7 7KLVVWXG\
9J &$7*7$&$777&&7777$&*WWDDWWDD&*7$*$$&**$$&&$7&*&*7*& 7KLVVWXG\
9J $7**77&&*77&7$&*WWDDWWDD&*7$$$$**$$$7*7$&$7**7$$$*&$$7&7*$&* 7KLVVWXG\
9J &&***&*$*&7&*DFWFJDJF&7**7$&&$&$*7&77$77**&*7*$7** 7KLVVWXG\
9J &$$7$$*$&7*7**7$&&$*JFWFJDJ77*777*$7&*&$&**77&&&$&$$7** 7KLVVWXG\
9J &&77$&$$&$*&&$&*&$*$$&*JJDWFF**&$***&$7*$$&*&***&777*$$*$& 7KLVVWXG\
9J *&&&*&*77&$7*&&&7*&&JJDWFF&*77&7*&*7**&7*77*7$$**777&* 7KLVVWXG\
9J *&7&$$&$**&7**&&77**7$FWFJDJ&7&$$&7*&&*$&*$*77*&7&* 7KLVVWXG\
9J &$$&7&*7&**&$*77*$*FWFJDJ7$&&$$**&&$*&&7*77*$*&$*&77*& 7KLVVWXG\
9J &*$7&*777$*7*$&*$*$7&$&*& 7KLVVWXG\
GV&DV) 7$$7$&*$&7&$&7$7$*7*&&$7&&7*&7*7&**$7$ 7KLVVWXG\
GV&DV5 7$$7$&*$&7&$&7$7$*&77&**&$*&$&&77&7&*7 7KLVVWXG\
GV.X) 7$$7$&*$&7&$&7$7$*&7$&**&$77***777*7&7 7KLVVWXG\
GV.X5 7$$7$&*$&7&$&7$7$**&77$*&77*7$*&&**$7* 7KLVVWXG\
*) &*7$7*&7*&$&*$&*77$$& 7KLVVWXG\

ϰ

*5 &7**$&$7&7*&77&7*$* 7KLVVWXG\
*5 *&$$7$*&7*$&7&&7* 7KLVVWXG\
.P) &*$7*&*$7&*$*&7$$77* 7KLVVWXG\
.P5 *7*$&*$*$7&$&*&$7&7* 7KLVVWXG\
9J¶5 &*777**7*&7&$*&7&$*$& 7KLVVWXG\
9J¶5 *7&*7$**7**7**7$7*&7$$& 7KLVVWXG\
8) &7$&&7*7$&*$7**&77$$* 7KLVVWXG\
P$)25 *$*$&**7*$$*$7&7&*7*&$$** 
P$5(9 *&77&*7$&&$&&$&&*$$&$&*7$ 
P&)25 $$***&7&*&7*$$$&7*7 
P&5(9 $&$*$$*7$&$&&*&&$**7 
$V9JIRU &$$&$7&$7*7&&$$*7&**$**7*$ 
$V9JUHY &77*$$*&777&*7*&7&77&&7&&* 
$V&3$)25 &777$&**$$$&*&7&*$$* 
$V&3$5(9 &&$7$*&$*$$7**$&&*7$$ƍ 
$VULE6IRU$$7&&77&&&*$$**$&$7*$$&&*
$VULE6UHY7$&*$$$&$$$7&&&$7&&7$$7&*$$*&
/RZHUFDVHOHWWHUVDUHLQWURGXFHGUHVWULFWLRQHQGRQXFOHDVHFOHDYDJHVLWHV


ϱ

7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
)RXQGHU &URVV /DUYDOSKHQRW\SHV
'V5HG'V5HG

ƃ
  
  
  
  
6XEWRWDOÁ 
Ƃ
  
  
  
  
  
  
  
6XEWRWDOÁ 
7RWDO  
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
3URJHQ\RIVLQJOH*PDOHRUIHPDOHRXWFURVVHGWRZLOGW\SHVRIWKHRSSRVLWHVH[
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ


7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
)RXQGHU &URVV /DUYDOSKHQRW\SHV
'V5HG'V5HG
'V5HG
NKZ
Ƃ
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
6XEWRWDO

  
ƃÁ%DWFK  
7RWDO   
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
3URJHQ\RIVLQJOH*IHPDOHRXWFURVVHGWRZLOGW\SHPDOHV
Á*PDOHVRXWFURVVHGWRZLOGW\SHIHPDOHV
ȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ

ϲ

7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
)RXQGHU &URVV
$GXOWSKHQRW\SHV
0DOHV )HPDOHV
'V5HGNKZ'V5HGNKZ'V5HG
PRVDLF 'V5HGNKZ'V5HG
NKZ'V5HGNKZ'V5HGNKZ'V5HG
PRVDLF 'V5HGNKZ'V5HG
NKZ
ƃ
   
   
   
   
7RWDOÁ  
Ƃ
        
        
        
     
        
      
        
7RWDOÁ         
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
3URJHQ\RIVLQJOH*PDOHRUIHPDOHRXWFURVVHGWRZLOGW\SHVRIWKHRSSRVLWHVH[
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ



ϳ


7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
)RXQGH
U &URVV
$GXOWSKHQRW\SHV
0DOHV )HPDOHV
'V5HGNKZ'V5HGNKZ'V5HG
PRVDLF
'V5HGNKZ'V5HG
NKZ'V5HGNKZ'V5HGNKZ'V5HG
PRVDLF
'V5HGNKZ'V5HG
NKZ
Ƃ
   1'    1'  
  1'    1'  
   1'     1'  
   1'    1'  
  1'    1'  
          
          
          
          
          
       
         
          
          
          
7RWDO         
ƃÁ%DWFK  
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
3URJHQ\RIVLQJOH*IHPDOHRXWFURVVHGWRZLOGW\SHPDOHV
Á*PDOHVRXWFURVVHGWRZLOGW\SHIHPDOHV
7KHILUVWFROXPQQXPEHUVDUHWKRVHPRVTXLWRHVZLWKPRVDLFLVPLQWKHH\H7KHVHFRQGFROXPQQXPEHUVDUHPRVTXLWRHVZLWKWKHµFRORUHG¶H\HSKHQRW\SHV
FRQVLVWHQWZLWKPRVDLFLVPLQWKHUHVWRIWKHERG\µ1'¶QRWGHWHUPLQHG
ȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ


ϴ

7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
&URVV7UDQVJHQLFSDUHQWQ/DUYDOSKHQRW\SHV
'V5HG'V5HG

'V5HGkhw-Ƃ  
'V5HGkhw-ƃ  
'V5HGkhw
+
ƂÁ 
'V5HGkhw
+
ƃÁ 
3URJHQ\RIEDWFKPDWHG*PDOHVRUIHPDOHVRXWFURVVHGWRZLOGW\SHVRIWKHRSSRVLWHVH[QLVWKHQXPEHU
RIWUDQVJHQLFIRXQGHUSDUHQWV
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ



7DEOH6*ODUYDOSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
&URVV7UDQVJHQLFSDUHQWQ/DUYDOSKHQRW\SHV
'V5HG'V5HG

'V5HGkhw-Ƃ  
'V5HGkhw-ƃ  
'V5HGkhw
+
ƂÁ 
'V5HGkhw
+
ƃÁ 
3URJHQ\RIEDWFKPDWHG*PDOHVRUIHPDOHVRXWFURVVHGWRZLOGW\SHVRIWKHRSSRVLWHVH[QLVWKHQXPEHU
RIWUDQVJHQLFIRXQGHUSDUHQWV
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ
7DEOH67DEOH6*ODUYDOSKHQRW\SHVRIOLQHVDQGEDWFK*LQWHUFURVVHV
 /DUYDOSKHQRW\SHV
&URVV 'V5HGkhw-'V5HG
khw-
/LQH  
/LQH  
*'V5HGSRVLWLYH'V5HGPDOHVDQGIHPDOHVZHUHLQWHUFURVVHGDVDVLQJOHEDWFKIRUHDFKOLQH
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG

ϵ



7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
/LQH &URVV)RXQGHU
$GXOWSKHQRW\SHV
*PDOHV *IHPDOHV
'V5HG
NKZ
'V5HG
NKZ
'V5HG
PRVDLF
'V5HG
NKZ
'V5HG
NKZ
'V5HG
NKZ
'V5HG
NKZ
'V5HG
PRVDLF
'V5HG
NKZ
'V5HG
NKZ

'V5HGkhw-Ƃ         
'V5HGkhw-ƃÁ            
'V5HGkhw+Ƃ          
'V5HGkhw+ƃ          
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
7KHILUVWFROXPQQXPEHUVDUHWKRVHPRVTXLWRHVZLWKPRVDLFLVPLQWKHH\H7KHVHFRQGFROXPQQXPEHUVDUHPRVTXLWRHVZLWKWKHµFRORUHG¶H\HSKHQRW\SHVFRQVLVWHQWZLWK
PRVDLFLVPLQWKHUHVWRIWKHERG\
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVp LQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ
 ȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ

ϭϬ







7DEOH6*DGXOWSKHQRW\SHVRIOLQH*RXWFURVVHVWRZLOGW\SHPRVTXLWRHV
/LQH &URVV)RXQGHU
$GXOWSKHQRW\SHV
*PDOHV *IHPDOHV
'V5HG
NKZ
'V5HG
NKZ
'V5HG
PRVDLF
'V5HG
NKZ
'V5HG
NKZ
'V5HG
NKZ
'V5HG
NKZ
'V5HG
PRVDLF
'V5HG
NKZ
'V5HG
NKZ

'V5HGkhw-ƂÁ         
'V5HGkhw-ƃ            
'V5HGkhw+Ƃ           
'V5HGkhw+ƃ          
6KDGHGFHOOVDUH*SURJHQ\SRVLWLYHIRU'V5HG'V5HG
7KHILUVWFROXPQQXPEHUVDUHWKRVHPRVTXLWRHVZLWKPRVDLFLVPLQWKHH\H7KHVHFRQGFROXPQQXPEHUVDUHPRVTXLWRHVZLWKWKHµFRORUHG¶H\HSKHQRW\SHVFRQVLVWHQWZLWK
PRVDLFLVPLQWKHUHVWRIWKHERG\
Áȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ
ȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVp LQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ
ȋ2DQDO\VHVVKRZVLJQLILFDQWGLIIHUHQFHVpLQ'V5HG'V5HGUDWLRVIURPUDQGRPVHJUHJDWLRQ

ϭϭ





)LJXUH60ROHFXODUFRQILUPDWLRQRIWKHSUHFLVHLQVHUWLRQVRIWKH$V0&5NKFDUJRLQWRWKHkhw ORFXV
6HTXHQFLQJRIWKHDQG¶HQG*)YJDQG8)*5MXQFWLRQDPSOLILFDWLRQIUDJPHQWVOHIWDQGULJKW
UHVSHFWLYHO\DPSOLILHGIURPWKH*IRXQGHUPDOHPRVTXLWRHVDQGORZHUVHTXHQFLQJWUDFHVUHYHDOHG
DSHUIHFWPDWFKWRWKHVHTXHQFHVIURP&DVPHGLDWHGFOHDYDJHDWWKHNKWDUJHWVLWHDQGSUHFLVHLQWHJUDWLRQRI
WKH$60&5NKFDUJR)LJXUHLQWRWKDWVLWH3RVLWLRQVRIWKHNKKRPRORJ\DUPVPDURRQER[HVJXLGH
51$WDUJHWLQJVHTXHQFHVEOXHER[HV3$0VLWHUHGDUURZKHDGDQGWKH3&5SULPHUV9JSULPHRUDQJH
ER[DQG8$6HTUHGER[DUHVKRZQ*HQRPLFQXFOHRWLGHVGHSLFWHGLQORZHUFDVHOHWWHUVLQGLFDWHUHVWULFWLRQ
HQGRQXFOHDVHHQ]\PHUHFRJQLWLRQVLWHVXVHGLQWKHFRQVWUXFWLRQRIWKH$V0&5NKFDUJR


ϭϮ



)LJXUH60XWDWHGVHTXHQFHVDWWKHNKWDUJHWVLWHLQVHOHFWHG*WUDQVJHQLFPRVTXLWRHV7KHWRS
VHTXHQFHLVWKHZLOGW\SHUHIHUHQFHVHTXHQFHDWWKHNKWDUJHWVLWHIURPDFRQWUROPRVTXLWR7KH3$0VHTXHQFH
LVVKDGHGLQSHDFKWKHQWWDUJHWLQJVHTXHQFHLVVKDGHGLQSXUSOHLQGHOVDUHVKDGHGLQSLQNDQGWKHJ51$
GLUHFWHGFOHDYDJHVLWHE\DWKLQYHUWLFDOZKLWHOLQH1+(-HYHQWVZHUHGHWHFWHGLQWKUHHGLIIHUHQWZKLWHH\HG
'V5HGPRVTXLWRHV'LIIHUHQWVHTXHQFHVZHUHLVRODWHGE\FORQLQJ3&5SURGXFWVJHQHUDWHGIURPWKHkhwORFXVLQ
OLQHVDQGVXSSRUWLQJWKHFRQFOXVLRQWKDWWKHVHPRVTXLWRHVDUHPRVDLFVFDUU\LQJDWOHDVWWZR
GLVWLQFW1+(-GLUHFWHGPXWDWLRQV,VRODWHLVGHULYHGIURPDGLUHFWVHTXHQFLQJUHDFWLRQRIDQ
DPSOLILFDWLRQSURGXFWFRQILUPLQJWKDWWKLVWUDQVJHQLFPRVTXLWRKDGRQO\DVLQJOH1+(-HYHQW,QFRQWUDVWWRWKH
RWKHUWZRFDVHVWKLVPXWDWLRQPDLQWDLQVWKHWUDQVODWLRQIUDPHEXWQRQHWKHOHVVUHVXOWVLQDZKLWHH\HSKHQRW\SH
VXSSRUWLQJWKHFRQFOXVLRQWKDWWKHGHOHWLRQRIDVLQJOHDPLQRDFLG<DQGVXEVWLWXWLRQRILWVQHLJKERU
*:HOLPLQDWHVRUJUHDWO\UHGXFHVN\QXUHQLQHK\GUR[\ODVHHQ]\PDWLFDFWLYLW\

ϭϯ











)LJXUH6*HQHDPSOLILFDWLRQRIWKHNKWDUJHWVLWHVLQVHOHFWHG*

IRXQGHUPRVTXLWRHV*HQRPLF'1$
SUHSDUHGIURPLQGLYLGXDOPDOHDQGIHPDOH*

IRXQGHUPRVTXLWRHVSRVLWLYHIRU'V5HGDQGZLWKZKLWHH\HV
'V5HG

kh
w-
FURVVHV7DEOHV6DQG6ZDVXVHGZLWKWKHJHQHVSHFLILFSULPHUVYJDQGYJ
7DEOH6WRDPSOLI\DSRUWLRQRIWKHkh
w
JHQH7KHGLDJQRVWLFIUDJPHQWDWEDVHSDLUVLQGLFDWHVDWOHDVWRQH
RIWKHFKURPRVRPHVKDGDkh
w
JHQHZLWKRXWWKHODUJHaNEWUDQVJHQHFDUJRLQVHUW7KHVHDOOHOHVPXVWEH
PXWDQWEHFDXVHWKHH\HSKHQRW\SHRIHDFKPRVTXLWRIURPZKLFKWKH'1$ZDVGHULYHGLVZKLWHkh
w-


¶&¶LVD
FRQWUROVDPSOHRI'1$IURPDZLOGW\SHPRVTXLWRµ0¶DUHWKHPROHFXODUZHLJKWPDUNHUV

DDĂůĞƐ&ĞŵĂůĞƐ
ϭϬ͘ϭ
ϭϬ͘Ϯ

ΕϳϱϰďƉ
ΕϳϱϰďƉ

ϭϰ

6XSSRUWLQJ0DWHULDOVDQG0HWKRGV
&RQVWUXFWLRQRIWKHvasa&DVFDVVHWWH
7KHDrosophila melanogastervasaJHQHDQGLWVAnopheles gambiaeRUWKRORJ$*$3ZHUHXVHGDV
%/$67VHDUFKTXHULHVWRLGHQWLI\WKH$67(JHQHDVWKHSXWDWLYHXQLTXHAnopheles stephensiRUWKRORJ
$ESJHQRPLFIUDJPHQWFRPSULVLQJWKHAnopheles stephensi vasaSURPRWHUDQGUHJXODWRU\VHTXHQFHV
SUHFHGLQJWKHSUHGLFWHGWUDQVODWLRQDOVWDUWVLWHZDVDPSOLILHGXVLQJROLJRQXFOHRWLGHVYJYJ)RXU'1$
IUDJPHQWVZHUHWKHQMRLQHGLQWKHIROORZLQJRUGHUWKHAn. stephensi vasaUHJXODWRU\IUDJPHQWDUHFRGHG
&DVJHQHFRGRQRSWLPL]HGIRUWUDQVODWLRQLQAnopheles stephensiS8&.DQ7$V&DVDPSOLILHGZLWKWKH
ROLJRQXFOHRWLGHVYJYJDESIUDJPHQWIROORZLQJWKHWUDQVODWLRQDOVWRSFRGRQ7$$RIWKH
$67(DPSOLILHGXVLQJWKHROLJRQXFOHRWLGHVYJWKHEDFNERQHRIWKHS%DF'V5HGSODVPLG
DPSOLILHGXVLQJWKHROLJRQXFOHRWLGHVYJYJ7KHVHIRXUDPSOLFRQVZHUHSXULILHGWUHDWHGZLWKWKH
UHVWULFWLRQHQ]\PHDpn,WRUHPRYHPHWK\ODWHG'1$DQGMRLQHGLQD*LEVRQDVVHPEO\1HZ(QJODQG%LRODEV
(6UHDFWLRQWRREWDLQWKHSODVPLGS9*S9*BS%DF'V5HGDWW%B$VWH9DVD&DV

&RQVWUXFWLRQRIWKH8$NKJ51$FDVVHWWH$ESIUDJPHQWVSDQQLQJWKHDQQRWDWHGAnopheles stephensi
8VQ51$JHQH$67(ZDVDPSOLILHGXVLQJWKHROLJRQXFOHRWLGHVYJYJDQGWKHDPSOLILFDWLRQ
SURGXFWZDVFORQHGLQWRS&57232XVLQJD7$7RSRLVRPHUDVH&ORQLQJ.LW,QYLWURJHQ7KH
UHVXOWLQJSODVPLGZDVXVHGDVDWHPSODWHIRUVLWHGLUHFWHGPXWDJHQHVLVXVLQJWKHROLJRQXFOHRWLGHVYJYJ
WKHUHE\VXEVWLWXWLQJWKH8WUDQVFULSWZLWKD[Bbs,UHVWULFWLRQVLWHOLQNHUIROORZHGE\WKHJ51$FRUHVHTXHQFH
7KHUHVXOWLQJSODVPLGS9*VHUYHVDVYHFWRUIRURQHVWHSLQVHUWLRQRIESJ51$JXLGHVHTXHQFHVWR
JHQHUDWHFRPSOHWHJ51$VXQGHUWKHFRQWURORIWKHAn. stephensi851$SRO\PHUDVH,,,SURPRWHUS9*
ZDVFXWZLWKWKHUHVWULFWLRQHQ]\PHBbs,DQGDOLQNHUJHQHUDWHGE\DQQHDOLQJWKHROLJRQXFOHRWLGHVYJYJ
ZDVOLJDWHGWRFORVHWKHJDSWKHUHVXOWLQJSODVPLGS9*H[SUHVVHVDJ51$WDUJHWLQJFOHDYDJHRIWKH
kynurenine hydroxylase-whitekhwFRGLQJVHTXHQFHDWWKHVHTXHQFH
³*$7**77&&*77&7$&***&$**´WKHEROGHG³***´FRUUHVSRQGVWRWKHHQFRGHGJO\FLQHRIWKH
khwJHQHSURGXFW

&RQVWUXFWLRQRIWKHKRPRORJ\DUPVEDFNERQHS9*7RPLQLPL]HWKHILQDOYHFWRUVL]HDPLQLPDO
ESEDFNERQHIUDJPHQWRIS8&ZDVDPSOLILHGXVLQJYJYJDQGIXVHGWRDNEJHQRPLFIUDJPHQW
IURPWKHAn. stephensikhwORFXVDPSOLILHGZLWKYJYJXVLQJ*LEVRQDVVHPEO\WR\LHOGWKHSODVPLG
S9*

&RQVWUXFWLRQRIWKHS$V0&5NKFRQVWUXFW7KHILQDOFRQVWUXFWS$V0&5NKZDVDVVHPEOHGXVLQJUHVWULFWLRQ
FORQLQJLQWZRVWHSV)LUVWDWKUHHZD\IXVLRQZDVJHQHUDWHGFRPSULVLQJDIUDJPHQWFRQWDLQLQJWKHS8&
SODVPLGEDFNERQHDQGWKHkhwKRPRORJ\DUPVZDVDPSOLILHGIURPS9*XVLQJYJYJZKLFKDGGHG
Pac,DQGBam+,UHVWULFWLRQHQ]\PHFXWVLWHVDWWKHHQGVDNE'1$IUDJPHQWLQFOXGLQJWKH[3'V5HG
H\HPDUNHUDQGDGXDOVLQJOHFKDLQVF)YDQWLERG\FDVVHWWHH[SUHVVLQJP&DQGP$DQGDPSOLILHG
ZLWKYJYJZKLFKDGGHGPac,DQG3sp;,VLWHVDWWKHHQGVDQGWKH8$NKJ51$FDVVHWWH
DPSOLILHGIURPS9*XVLQJYJYJZKLFKDGGHGPsp;,DQGBam+,VLWHVDWWKHHQGV(DFKRIWKH
WKUHHIUDJPHQWVZDVFXWZLWKWKHUHVSHFWLYHUHVWULFWLRQHQ]\PHVIROORZHGE\WUHDWPHQWZLWKDpn,WRUHPRYHWKH
PHWK\ODWHGWHPSODWHVDQGOLJDWHGWRJHWKHUWRREWDLQWKHLQWHUPHGLDWHSODVPLGS9*,QWKHVHFRQGVWHSWKH
WZRDERYHIUDJPHQWVZHUHMRLQHG7KHYDVD&DVFDVVHWWHZDVDPSOLILHGIURPS9*XVLQJYJYJ
ZKLFKDGGHGPsp;,VLWHVDWERWKHQGVDQGWKHREWDLQHGDPSOLFRQZDVFXWZLWKPsp;,DQGDpn,DQGWKHQ
OLJDWHGWRS9*OLQHDUL]HGZLWKPsp;,DQGWUHDWHGZLWKFDOILQWHVWLQDODONDOLQHSKRVSKDWDVHWRREWDLQWKH
ILQDOFRQVWUXFW

5HDJHQWVDOODPSOLILFDWLRQVWHSVZHUHSHUIRUPHGXVLQJ3KXVLRQ+LJK)LGHOLW\'1$3RO\PHUDVHIRUIUDJPHQWV
NE1HZ(QJODQG%LRODEV&DW06RU4+LJK)LGHOLW\'1$3RO\PHUDVHIRUIUDJPHQWV!NE1HZ
(QJODQG%LRODEV&DW067KHHQ]\PHVPHQWLRQHGZHUHSXUFKDVHGIURP1HZ(QJODQG%LRODEVBam+,

ϭϱ

+)56Bbs,56Dpn,56Pac,56Psp;,56FDOILQWHVWLQDODONDOLQH
SKRVSKDWDVH06

5HIHUHQFHV
,VDDFV$7/L)-DVLQVNLHQH1&KHQ;1LUPDOD;0DULQRWWL29LQHW]-0DQG-DPHV$$
(QJLQHHUHGUHVLVWDQFHWRPlasmodium falciparum GHYHORSPHQW LQWUDQVJHQLFAnopheles
stephensiPLoS Pathogens30,'30&,'30&
Ϯ͘1LUPDOD;0DULQRWWL26DQGRYDO-03KLQ6*DNKDU6-DVLQVNLHQH1DQG-DPHV$$
)XQFWLRQDOFKDUDFWHUL]DWLRQRIWKHSURPRWHURIWKHYLWHOORJHQLQJHQHAsVg1RIWKHPDODULDYHFWRU
Anopheles stephensi. Insect. Biochem. Molec. Biol.,.30,'
,VDDFV$7-DVLQVNLHQH17UHWLDNRY07KLHU\,=HWWRU$%RXUJRXLQ&DQG-DPHV$$
7UDQVJHQLFAnopheles stephensiFRH[SUHVVLQJVLQJOHFKDLQDQWLERGLHVUHVLVWPlasmodium falciparum
GHYHORSPHQWProc. Natl. Acad. Sci.USA((30,'PNAS PLUS

&KHQ;0DULQRWWL2:KLWPDQ/-DVLQVNLHQH15RPDQV3DQG-DPHV$$7KHAnopheles
gambiaeYLWHOORJHQLQJHQHVGT2SURPRWHUGLUHFWVSHUVLVWHQWDFFXPXODWLRQRIDUHSRUWHUJHQHSURGXFW
LQWUDQVJHQLFAnopheles stephensiIROORZLQJPXOWLSOHEORRGPHDOVAm. J. Trop. Med. Hygiene
30,'
3DSDWKDQRV3$:LQGELFKOHU10HQLFKHOOL0%XUW$DQG&ULVDQWL$7KHYDVDUHJXODWRU\
UHJLRQPHGLDWHVJHUPOLQHH[SUHVVLRQDQGPDWHUQDOWUDQVPLVVLRQRISURWHLQVLQWKHPDODULDPRVTXLWR
$QRSKHOHVJDPELDHDYHUVDWLOHWRROIRUJHQHWLFFRQWUROVWUDWHJLHV%0&0RO%LRO-XOGRL
30,'
