CRISPR/Cas9 and Active Genetics

Valentino Gantz

Cas9

• Nuclease: Cas9 �

CRISPR/Cas9 technology

• Genomic Target : N20 NGG �

...TGTGGGTTTTGGACACTGGAACCGTGGGCATCG...

...ACACCCAAAACCTGTGACCTTGGCACCCGTAGG...˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙Genomic DNA

Cleavage Site

PAM

GGUUUUGGACACUGGAACCG

gRNA

• gRNA : N20 gRNA sequence �

Cas9 gRNA

Intact sister chromosome B

Intact chromosome A

CRISPR/Cas9 technology

Cas9 gRNA

Intact sister chromosome B

Double stranded DNA break repair

Chromosome A with double stranded break

Non-homologous end-joining (NHEJ)

Error prone repair: indels

Homology Directed Repair (HDR)

Repair by copying from Chromosome B

CRISPR/Cas9 technology

The Mutagenic Chain Reaction (MCR): Chromosomal Insertion

HA HACas9 gRNA

Cas9 gRNACas9 gRNA

MCR conversion of sister allele

Cas9 gRNA

Cas9 gRNACas9 gRNA

MCR conversion of sister allele

x

mutant wild-type

Mendelian vs MCR inheritance

x

mutant wild-type

8

50% allele inheritance 100% wild type

Mendelian vs MCR inheritance

x

mutant wild-type

9

x

mutant wild-type

50% allele inheritance 100% wild type 100% mutant

100% allele inheritance

Targeting yellow with an MCR

yellow-MCR inheritance

y- ♂ y- ♀ mosaic ♀ ♂ ♀ F2

0 40 0 50 1 91

214 203 11 2 6 436 y-MCR ♀ x ♂ wt

y-MCR ♂ x ♀ wt

tot. ♀/2 - non-MCR ♀ tot. ♀/2

= 96%wt ➛ MCR

conversion rate

Logistic Spread of an MCR Element

Alle

le F

ract

ion

Generation

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1 2 3 4 5 6 7 8 9 10

MCR

gRNA-3 gRNA-2

Cas9 gRNA-1

MCR

ERACR eye-DsRed

Element for Reversing the Autocatalytic Chain Reaction (ERACR)

Element for Reversing the Autocatalytic Chain Reaction (ERACR)

ERACR

ERACR

gRNA-3 gRNA-2

eye-DsRed

gRNA-3 gRNA-2

eye-DsRed

Alle

le F

ract

ion

Generation

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

ERACR

Modeling MCR versus ERACR

MCR

Potential applications

MALARIA

Blood meal Reduced malaria transmission

AgCP [effector]Cas9 gRNA

“If coupled with a mechanism for gene spread…malaria-resistance transgenes could become a self-sustaining disease control tool.”

Isaacs el al. 2012

Potential application: Malaria

Numbers Transmission Rate Conversion

Rate Insert Size

3798 98.9% 97.9% 8 kb

3804 99.5% 98.8% 17 kb

MCRs efficiently spread genes in flies and mosquitoes

(James Lab - UCI; Vinetz Lab - UCSD)

Potential application: HIV

Entry

Retro transcription

Integration Transcription

Assembly

Exit

CD4+ T Cell

genome

Entry

Retro transcription

Cas9-mediatedintegration

xSpecific

sequence

Uninfected Infected

Potential application: HIV

Entry

Retro transcription

Cas9-mediatedintegration

Assembly

Viral genes transcription

andtranslation

Exit

x

Uninfected Infected

Potential application: HIV

D. melanogaster poses NO threat to human health or agriculture.

Nonetheless, because MCRs have the potential to rapidly disperse in local or worldwide populations we followed a stringent institutionally approved confinement protocol:

•  3 levels of physical containment.

•  All flies killed before phenotypic analysis.

•  All fly work performed in an ACL2 insectary in a secure barrier containment facility (UCSD).

•  All mosquito work performed in an ACL2 insectary (UCI) under an approved protocol.

MCR flies

Safety considerations for laboratory experiments

New Covenant
Jesse Bier

Nobody tells the flowers what to do,they grow the way they please.

They don't take orders from me and you—nor do the grasses or trees.

But that was long ago,before the age of science.

Now that we know what we know,we splice them to compliance.

Soon trees must do what they are told,according to special genes.

Nothing the same as of old—and grasses, flowers know what that means.

Next to come are you and meafter the flowers, trees and grasses.But: Who will decide and oversee

newfounded families, permanent classes?