El 12 de mayo de 2017 celebramos en la Fundación Ramó Areces una jornada con IS Global y Unitaid sobre enfermedades transmitidas por vectores, como la malaria, entre otras.

1. Andrea Crisanti
Imperial College London
Gene drive technology for malaria vector control
in Anopheles gambiae

2. Gene drive can be engineered to:
Population replacement Population suppression/elimination
Replace a pathogen-susceptible mosquitoes
population with pathogen-resistant insects
Induce population suppression or elimination
of wild type mosquitoes

3. Population suppression or elimination of wild type mosquitoes
Skewing
Sex Ratio
Disrupting
Female Fertility
♀
A
A
X
♂ ♂ ♀
Homing
~100% inheritance
of the transgene
♂ ♂
♂
Y
X
♀
X
X
X
Sex Ratio Distortion
>50% ♂ : <50% ♀
Recessive
female-sterility
phenotype
Endonuclease
♂
A
A
♀
♀♀

4. Putative Female Fertility Genes
Gene Ortholog in Drosophila Putative function
AGAP007280 nudel Embryonic patterning
AGAP005958 yellow-g Egg chorion formation
AGAP011377 None identified Chorion maturation

5. CRISPRh gene drives confer strong unintended fertility effects
in females
AGAP011377
AGAP005958
AGAP007280
Females heterozygous for the
gene KO are fertile, but when
they have the CRISPRh homing
construct, they are sterile or
semi-sterile.
Males are fertile with the KO
and with the CRISPRh construct
Heterozygous ♀ Heterozygous ♂
larvae
♀ ♂

6. Vasa-driven Cas9may have leaky expression
Germline
Soma
Gene required here
Homing happens here
vasa
vasa?

7. CRISPR-based gene drive spreads in a cage population
Cage 1
Cage 2
predicted
This is the first time a synthetic gene drive
has been shown to target a natural gene
and spread into a population
Popn size=600

8. Development of resistance to gene drive
• Target site variants could be resistant to endonuclease cleavage and thus not
susceptible to drive
• Such variant alleles confer strong fitness advantage and will spread rapidly at the
expense of the drive allele if they still encode a functional gene
• Variant alleles might be pre-existing in the population OR might be induced by the
mutational activity of the endonuclease

9. 0%
25%
50%
75%
100%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
FrequencyofCRISPRhallele(%)
Generation
Change in CRISPRh allele frequency over time
low frequency mutant alleles
in-frame and frame shift
pooled sequencing
high frequency mutant alleles
majority in-frame
pooled sequencing
Encode functional allele
Resistant to further cleavage
Phenotypic analysis
individual sequencing
Select mutant alleles
all in-frame
• Monitoring of Resistance to HEGs

10. Gene Drive Challenges
DNA off-targets Cell/Tissue specificity
Genetic resistance Safety and Logistic

11. • Alternative germline-restricted promoters to
improve performance of homing constructs
No. larvae% RFP+
Homing rates from nanos and zpg
promoters are very high
Improved fertility in heterozygous
females
mean mean

12. Anopheles gambiae 1000 genomes project
- Existing Variation at Target Site in Natural Populations
http://www.sanger.ac.uk/science/tools/ag1000g

13. AGAP004734 -PAX-interacting protein 1
Ultraconserved regions (>18bp, 100% identity between 21 An. gambiae species)
Alternating exons
Alternating exons
Current HEG site  which mutated in population cage experiments
Proposed CRISPR target site

14. The N-terminal part of Exon 5 is not only conserved on protein level it has a long
ultra-conserved DNA stretch with no known variants (in red; source: 1000 Genomes
project)
Ultra-conserved DNA stretch within Exon 5
Exon5

15. Gene disruption by homology-directed repair (HDR) at a conserved site within Exon5
causes recessive lethality
modified from Hammond et al., 2016
mutation is recessive lethal (all hdrGFP/hdrGFP individuals die
during larval devlopment)

16. Homozygous individuals hatch, but die before adulthood
If HR is 93% we would expect survival of
13% heterozygous (CRISPRh/+) and 0.49% RFP- Larvae (+/+)
~93%
12.5%
0
200
400
600
Ad u lts
R F P + L a r v a e
R F P - L a r v a e
E g g s
c o u n t
0.24%
CRISPRh/+ x CRISPRh/+

17. Dynamics of a CRISPRh x CRSPRh population over several generations
The number of adults does not change significantly over
generations
0
200
400
600
800
Ad u lt s ta r tin g p o p u la tio n G 1
E g g s G 2
L a r v a e G 2
R F P + L a r v a e G 2
Ad u lt G 2
E g g s G 3
L a r v a e G 3
R F P + L a r v a e G 3
Ad u lt G 3
E g g s G 4
R F P + L a r v a e G 4
L a r v a e G 4
Ad u lt G 4
c o u n t
(G1, G2, G3, G4)
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
generation 1 genertaion 2 generation 3 genration 4
WT population
reproductive females eggs larvae adult

18. The genetic drive at the selected lethal locus has ablated the biotic potential: the
ability of mosquitoes to occupy their ecological niche
Predicted
Experimental determination

19. x
Stages of Testing…

20. Mosquito Confined Release Facility
1 small and 1 LARGE
climatic chamber
 Stringent biosafety measures for the contained housing of gene drive technologies
- Physical, Environmental and Biological Containment (level BSL2/ACL2+)
 Mimic natural environments (temperature, humidity and light cycle) to promote
natural behaviours (assortative mating and male swarming) and evaluate fitness
and competitiveness of GM strains
 Rear large number of mosquitoes (consecutive defined or overlapping generations)
 Accurate validation of modelling predictions of gene drive dynamics
 High statistical power to detect rare events
Small vs Large cage survival

21. Andrea Crisanti
Tony Nolan
Galizi Roberto
Silke Fuchs
Federica Bernardini
Andrew Hammond
Roya Haghighat-Khah
Alekos Simoni
Kyros Kirou
Chrysanthi Taxiarchi
Antonios Kriezis
Nace Kranjc
Matthew Gribble
Christopher Bamikole
Dario Meacci
Giulia Morselli
Ann Hall
Mariana Reis Wunderlich
Louise Marston
Lucy Collins
Clelia Supparo
Philippos Papathanos
Nikolay Windbichler
Greta Immobile Molaro
Ruth Muller
Paola Pollegioni
Laura Chiti
Roxana Minuz
Marco Bruttini