enhancing biodiversity through genetic rescue for endangered

and extinct species

Ryan Phelan

Animal Biotech Summit

September 22, 2016

Classic “Genetic Rescue”

Western Mountain Lion

Florida Panther Historic range

Present range

Genetic Rescue to Save a Species

Revive & Restore’s Black-footed Ferret Project

genetic rescue for endangered and extinct species

Black-footed Ferret

Once Thought to be Extinct . . . Now

Endangered

Presumed extinct

by 1950

Rediscovered in

Wyoming in 1981

(with 18 individuals)

EXTINCT

Restoring a Healthy Ecosystem

Disease Prevents Recovery

Asian Sylvatic Plague Yersinia Pestis

Extinction vortex

Genetic

rescue

DNA from 2

cryopreserved ferret

specimens

DNA from 2 living specimens

Founders

1985 Wild Meeteetse Population

(Generation 0-1)

Present Captive-bred Population (approx. 15-20 generations

descended)

BFF Study Demography

• Ferrets Captured from

Meeteetse

• Only a portion successfully

breed

• >8,000 kits born the next 30

years

• All are related like

cousins/siblings

• Artificial Insemination used for

first genetic rescue efforts

Individuals selected for whole genome comparison

SDFZ cell lines

Whole genome comparison goals:

• Assess changes in genetic diversity

• Assess genetic rescue efforts

• Assess genetic rescue potential of

SDFZ cell lines

Conclusions:

• Genetic diversity erosion (decrease

in heterozygosity and decrease in

unique variation) observed between

1985 individuals and Cheerio

• Balboa’s genetic diversity was

intermediate between 1985 and

Cheerio’s values

• 1985 individuals were distinct from

Cheerio’s variation (which is

descended from the founders)

ID 85 W2094 Willa

Balboa Cheerio

Novel Genetic Variation for Ferrets

Historic range Last breeding rang

Restoring Lost

Diversity

1985 2016 1955

Pop

ula

tio

n S

ize

Experimental Design Meeteetse Genomes Extinct Populations’ Genomes

SNP

SNP shared by extinct populations, but absent

in Meeteetse Engineered “outbreeding” genome

Engineering Disease

Resistance

Skin Cells

Macrophage Cells

Modified Receptors

Testing genome-edits

in a petri dish

Genetic Rescue - The 5 Stages

In Vitro

CTCCCCCC

ATCCTTTC

TTCTCCTT

CTAGCCTC

CTCCACAG

TCGAAGCT

GGTGCAGG

AACAGGAT

TAGCCCAC

ACCTTGCC

ATCTTCTC

ACTCCACC

TTGCTGGT

GTCTCCTC

CATCCTAG

GGGCCATC

AACTTTAT

CACAACCG

CCATTAAT

ATAAAACC

CCCCGCCC

TTTCACAA

TACCAAAC

TCCCCTAT

TCGTATGA

TCGGTCCT

CATTACCG

CCGTCCTC

CTTCTCCT

ATCCCTCC

CAGTCCTT

GCCGCTGG

CATCACAA

TATTACTC

ACAG

In Silico

Genome sequencing

In Vitro

Genome editing

In Vivo

Genome-edited progeny

Ex Situ

Captive Breeding & Care

In Situ

Return to the wild

Genetic Rescue Candidates

• Black-Footed Ferret

• Hawaiian Crow

• Millerbird

• Grey Ox

• Red Wolf

• African Wild Ass

• Hunter’s Antelope

• Northern Hairy-Nosed

Wombat

• Christmas Island

Shrew

• Exuma Rock Iguana

GENETIC BOTTLENECK

• Scarlet Honeycreeper

• Kaua’i Creeper

• Mangrove Finch

• Layman Duck

• Baxter’s Toad

• Fleischmann’s Robber Frog

DISEASE-CHALLENGED

INVASIVE SPECIES PRESSURE • Amsterdam Albatross

• Fiji Crested Iguana

• Ashy Storm-Petrel

Advancing Biotech for Wildlife

Conservation

genetic rescue for endangered and extinct

species

21st Century Conservation Challenges

• Disease

• Invasive Species

• Climate Change

• Extinction

…need 21st century solutions.

“We’re going to

have to science

the shit out of

this.”

Revive & Restore: Catalyst and Facilitator

Stamping Out Mosquitoes in Hawaii

Can new technology stop avian malaria from

driving Hawaii’s native birds to extinction?

In Hawaii native birds survive

avian malaria only above 4,000 feet.

‘Akeke‘e Robby Kohley

‘Anianiau Robby Kohley

Kaua‘i ‘Amakihi Robby Kohley

Maui Parrotbill (Kiwikiu) Robby Kohley

‘I‘iwi Robby Kohley

Maui ‘Alauahio Robby Kohley

Hawai‘i ‘Ākepa Robby Kohley

Hawai‘i Creeper Chris Farmer

Palila Keith Swindle

‘Akikiki Robby Kohley

‘Akiapōlā‘au Jack Jeffrey

‘Ākohekohe Robby Kohley

Genetic Rescue

Can new genomic tools solve conservation

problems such as exotic wildlife diseases and

destructive invasive species?

Less than 5% of land mass

80% of extinctions since 1500

40% of endangered species

ISLANDS REPRESENT

Cause of Island Extinctions: Invasive Species

Hawaii

Living

Endemic Bird Species

Invasives: • Destroy habitat • Kill young • Spread disease

Extinct

New Zealand

0

22.5

45

67.5

90

112.5

Cause of extinction

Bird Extinctions

Humans

Invasives

Guam

10 of 12 forest birds extinct in 60 years

Ecological functions have stopped

Current Method Limitations

• Must reach every rodent

territory

• Not species specific

(collateral death)

• Animal welfare issues

• Human interference

• Negative perception of

toxicants

CRISPR/Cas9 Gene Drive

“…A cynical move is underway to promote a new, powerful, and troubling technology known as “gene drives” for use in conservation. This is not just your everyday genetic modification, known as “GMO”; it is a radical new technology, which creates “mutagenic chain reactions”… using gene-drive technology for conservation is a ruse to gain public acceptance and regulatory cover.”

CALLS UPON the Director General and Commissions with urgency to assess the implications of Gene Drives and related techniques and their potential impacts on the conservation and sustainable use of biological diversity as well as equitable sharing of benefits arising from genetic resources, in order to develop IUCN guidance on this topic, while refraining from supporting or endorsing research, including field trials, into the use of gene drives for conservation or other purposes until this assessment has been undertaken.

“The likely benefits of gene

drives for basic and applied research are significant and justify proceeding with laboratory research and highly-controlled field trials.”

ENGAGE THE PUBLIC — EARLY AND OFTEN

Align research with public values -Clearly articulate benefits and risks Clarify vision: • Restore biodiversity • Increase bioabundance • Self-managing populations

enhancing biodiversity through genetic rescue for endangered

and extinct species

www.reviverestore.org