Using CRISPR-Cas9 to produce double sex of Anopheles gambiae to reduce in the population, and decrease in malaria transfection.

1. CRISPR-cas9 on eradicating pests
Teoh zhi lingYong shu wenLiew chen hau ASwint
To study CRISPR–Cas9 gene drive targeting doublesex
causes complete population suppression in caged
Anopheles gambiae mosquitoes

2. Malaria PATHOLOGY
● Sporogony and sporozoite production in
mosquito.
● A female anopheline mosquito injects
the “sporozoites” while taking the blood
meal
● Parasite multiplies in RBC, RBC burst
and spread through bloodstream.
● Symptoms: Fever (>38’C), shivering,
headaches, vomiting, diarrhoea, muscle
& joint pains.
*Parasite matures in the liver*

3. What makes male and Female A.giambae different?..
● Clustered Regularly Interspaced
Short Palindromic Repeats
● Cas 9 (produced by bacteria) is
used to cut the targeted site by
gRNA.
● Low cost-gene editor
● Anopheles gambiae encodes two alternatively spliced transcripts, dsx-female and dsx-male.
● The female transcript contains exon 5 which is a highly conserved sequence in all anopheles
mosquitoes.
● The male specific isoform contains an additional domain at the C terminus that is transcribed
as a noncoding 3’ untranslated region in females

4. Germline promoter
Reporter gene
Left recombinase
target site
Right recombinase target site
Transcription unit
U6 promoter
Guide
RNA
Disrupted
exon 5
Recombinase-mediated
cassette exchange:
● Replace 3xP3::GFP
transcription unit
with a dsxFCRISPRh
gene drive
3xP3::GFP transcription unit
Flanking
sequence
Integrase
target site
HDR knockout construct:
● Recognize exon 5 &
corresponding target
locus.
● Induce DSB

5. GFP gene
knock in
1. Incorporate
dsxFCRISPRh gene
drive into target
locus.
2. Transformant
confirmed by
swapping of GFP
with RFP.
Experimental outline
1. Cas 9 with designed
gRNA cleave intron
4-exon 5.
2. Insert eGFP by HDR
3. Disruption of exon 5
4. Confirmed by PCR &
gene sequencing
Cross
breeding
1. Knock-in of
eGRP gene is
identified by
PCR.
1. Heterozygous
individuals crossed
with heterozygous.
2. Produce WT,
homozygous of
having eGRP, and
heterozygous.
Recombinase-
mediated casette
exchange (RMCE)
Selection
Is dsx a suitable
target for a gene
drive?
1
2
3
4

6. Results
Reproductive phenotype
of mutant dsxF gene Morphology of dsxf -/-
Female mosquito carried clasper
(male specific characteristics),
resulting an insersex mosquito
Could not feed on blood and failed
to produce eggsFemale mosquito with dsxF-/- has
no ability to produce larval progeny.

7. Assessment of dsx gene drive in caged insects
It is shown that
heterozygous male has
95.9% ± 1.1%
heterozygous female has
99.4% ± 0.5%of
transmission rate of dsxF
CRISPR to the progeny.

8. Gene drive dsxFCRISPRh
Anopheles mosquitoes with boosted
immune Y-drive
Current Development
● High inheritance bias.
● Heterozygous are fully
fertile.
● Homozygous females are
sterile and unable to bite.
● No evidence for
nuclease-resistant
functional variants.
● Suppressed
Plasmodium parasites.
● Trait found in ±90% of
the 1st generation,
remained through 10
generations.
● Targets haplo-sufficient
fertility genes
● Introduce sex distorter
on the Y chromosome in
the form of nuclease.
● Shreds X chromosomes.

9. Challenges and
Limitations
competition for resources or
mating success
Environmental concerns
by society
difficulties in sustaining
controlled practices
development of resistance
to nuclease-based gene
drive

10. ● Female mosquitoes genetically engineered with
dsxFCRISPRh gene drive
● Disabled blood meal in female mosquito progeny
➢ Progeny key phenotype: Clasper
● dsxF preferred method as high inheritance rate, fully
fertile heterozygotes, homozygous females are sterile
● Other developments include boosted immunity
mosquitoes, and “y-drive”
● Challenges include competition for resources and
mating success, controlled practices unsustainable,
resistance to nuclease-based gene drive, and
complexity of transmission dynamics
CONCLUSIONS

11. References
1. Kyrou, Kyros, Hammond, Andrew, Galizi, Roberto, Kranjc, Nace, Burt, Austin,
Beaghton, Andrea, Nolan, Tony, Crisanti, Andrea 2018, “ A CRISPR–Cas9 gene
drive targeting doublesex causes complete population suppression in caged
Anopheles gambiae mosquitoes”, Nature Biotechnology, vol 36, no. 11,
viewed on 20 August 2020,
<https://www.researchgate.net/publication/327848989_A_CRISPR-Cas9_gen
e_drive_targeting_doublesex_causes_complete_population_suppression_in_
caged_Anopheles_gambiae_mosquitoes>
2. National Institue of Health 2017, “Engineering malaria resistance in
mosquitoes”, NIH Research Matters. Viewed on 24 September 2020,
<https://www.nih.gov/news-events/nih-research-matters/engineering-malari
a-resistance-mosquitoes>