Genetic Modification For Potential Biocontrol

Aquatic Invaders Summit

St Cloud, MNSt Cloud, MN

05-October, 2016

Chris Merkescmerkes@usgs.gov(608) 781-6316

What is genetic control?

Genetic control is the exploitation of genetics to get the organism to control ITSELFITSELForganism to control ITSELFITSELF.(letting nature do the work for us) 

3 Types of genetic control:

Y Y l l Y‐Y male release

RNA interference

CRISPR gene drives

Y-Y male release

PREMISE: Skew sex ratios so that there are only males…Then the invader cannot reproduceThen the invader cannot reproduce.

Won’t work for all invasive species…

Depends on XY sex determination anddetermination, and some other factors.

“Normal” Sex Determination

Humans have 22 pairsHumans have 22 pairs of “autosomes”…

… and 1 pair of“sex chromosomes”sex chromosomes

“X”  “Y” – This person is male

“X” “X” – This person is femaleX    X   This person is female

Other types of Sex Determination

ZW Sex Determination• Chickens, Komodo Dragons, Giant 

River Prawn, Moths and Butterflies, Others…

X0 Sex Determination• Grasshoppers, Crickets, Cockroaches, 

Others…

Haplodiploid Sex Determination Haplodiploid Sex Determination• Bees, Ants, Wasps, Thrips, Others…

Temperature‐DependentSex Determination• Reptiles, Possibly others…

Y-Y Male Release to control a population:How it worksHow it works

x y

Normal male mated 

xx

x

x

y

xx

xx xy

xy 50% female

50% lwith normal female: x xx xy 50% male

Super male mated 

x yx

y

xy xy 100% malep

with normal female: x xy xy 100% male

Y-Y Male Release to control a population:How it’s madeHow it s made

X‐Y genetically male fish will develop into phenotypically female if exposed to estradiol during development

These X‐Y egg producers can then be mated with malesgg p

x y

Normal male mated 

xx

x

y

y

xx

xy yy

xy 25% female 50% normal male

25% lwith feminized males: y xy yy 25% super male

Y-Y Male Release to control a population:ConsiderationsConsiderations

Deal Breakers: Sex must be determined genetically with X‐Y systemSex must be determined genetically with X Y system

with no environmental influence Individuals must not be able to spontaneously 

reverse sexes Aquaculture methods must be possible

Deal Makers:Deal Makers: Low density target populations Short‐lived species Harvestable

Y-Y Male Release to control a population:ConsiderationsConsiderations

Other Considerations:

Drop it like its hot – Reversible

l l Must release many super males

Must continue for yearsy(dependent on species)

Releasing sex‐hormone‐treated fish Releasing sex‐hormone‐treated fish(FDA regulation)

Y-Y Male Release to control a population:ImplementationImplementation

1. Develop Y‐Y super males

2. Culture super males for release

3. Harvest target species

4. Release marked super malesp

5. Harvest target species (releasing recaptured super males)(releasing recaptured super males)

6. Repeat – Culture/Harvest/Release

RNA interference (RNAi)

PREMISE: Trick the organism into “thinking” important genes are unimportant on the cellular levelgenes are unimportant on the cellular level.

Use the organism’s own Use the organism s own molecular machinery to shut down cells.

Specific to DNA sequence beyond just species, but y j p ,targets genes of that species.

The Central Dogma of Biology

DNA ‐> RNA ‐> Protein

XX

RNA interference targets RNA molecules forRNA interference targets RNA molecules for destruction thus preventing protein from being made

RNA interference

RNAi tricks the cell into destroying its y gown mRNA

RISC – RNA Induced Silencing Complex

Small d bl d ddouble‐stranded RNA molecules guide RISC in what mRNA to slice upmRNA to slice up

We choose very specifically what genes to turn offWe provide the small dsRNA, and the target individuals do the rest

RNA interference: Delivery Mechanism

Microparticle: Filled with interfering dsRNA Filled with interfering dsRNA May require significant dose Apply as needed – degrades over time

Food source: Genetically modified plankton/etc produces interfering RNA Establish GMO population – Inoculates water body from invasionp p y Risk of escapement Potentially difficult to reverse

Currently in use by agriculture – GMO corn resistant to corn root wormNo off‐target effects are known – Still needs further study

RNA interference: Research Required

Identify genes to target (may require sequencing)

Design interfering RNAs

Confirm interfering RNAs alter gene expression (in vitro)

Confirm altered gene expression lethality (in vitro)

Develop delivery mechanism

Lab trials

Field trials

Regulatory approval – Could be FDA and EPA

CRISPR gene drives

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats – A method for editing genomes.p g g

What’s so special about CRISPR?

Compared to previous genome editing methods:

Faster Easier and Cheaper Faster, Easier, and Cheaper

• Days instead of weeks or months

• Sequence based targeting• Sequence‐based targeting

• Hundreds instead of Thousands of $$$

More reliable targeting More reliable targeting

• 95‐98% fidelity

Fewer off target affects Fewer off‐target affects

• Less chance of unintended consequences

What’s so special about CRISPR?

Originally discovered as a bacterial defense against virusesg

• Chops up viral DNA

First human trials approved this yearpp y

• Cured Retinitis Pigmentosa

• Cured Leukemia

• Aids cure on the way

• Many genetic diseases curableMany genetic diseases curable

Malaria Resistant Mosquitos in development

How CRISPR can help with invasive species

Gene Drive: A gene that is guaranteed to pass on to all offspring.p g

Can be:

Lethal alleles

Female sterility allelesy

Modified function alleles

Considerations in using CRISPR gene drives to control an invasive speciesdrives to control an invasive species

Sexual reproduction required

Requires releasing potentially many genetically modified individuals

Not reversible

Risk for escapement

Less risky with “Daisy‐chain” drive

Research required to use CRISPR gene drives to control invasivesdrives to control invasives

Statistical modeling of the problem

In depth understanding of target species genome

• Beyond just the sequence

Design strategic CRISPRs

Develop genetically modified organisms for release

• May be a continual need

Lab trials

Field trials

Regulatory approval

Ethical Considerations

Genetic modification has been around a long time

• Crops, Dogs, Race horses

Incredibly powerful tool

What we “can do” vs. what we “should do”?

Public engagement

• Interested parties

• Scientists

• Politicians

• Public at large

Chris Merkes – cmerkes@usgs.govUSGS – Upper Midwest Environmental Sciences CenterUSGS  Upper Midwest Environmental Sciences CenterLa Crosse, Wisconsin(608) 781‐6316