FOI 240-1718

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Genome Engineering

David Tscharke, John Curtin School of Medical Research, ANU

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 A Brief History of Genetic Modification

• The first GMO was made in 1973

› Bacteria carrying DNA of another species

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• Most manipulation of DNA has been done using short

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fragments in test tubes and bacterial systems

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• This DNA can be transferred to human / animal cells to

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add something new to their genome

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› Naked DNA or packaged into a virus ‘vector’ for delivery
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› Insertion is random → unexpected consequences

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› Could be inserted into fertilized ovum to make an animal

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• First targeted transgenic animal (mouse) made in 1987

› Genes could be removed or changed in an animal

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 A brief history of genetic modification II

• Polymerase chain reaction (PCR) and chemical

synthesis of DNA made things faster

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• Methods like polymerase chain reaction (PCR) and

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chemical synthesis of DNA → synthetic biology

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› Still a long way off making mammals

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• RNAi allowed genes to be turned down

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› Has to be a constantly present
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• Achieving a desired genetic change - even in cells in

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tissue culture remained practically impossible

› This is the first step towards therapeutic genome modification

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Gene therapy – could only add new DNA

Kaufmann KB et al, EMBO Mol Med,

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2014
 Key limitations
• Most cutting and modification done in vitro not in cells
› Have to get DNA out of cells and back in again
› Limits the length that can be easily modified

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• The enzymes used to cut had defined recognition sites

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› E.g. GAATTC

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› They rarely cut exactly where you want

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› They cut at many other places

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• PCR and synthetic DNA methods were helping
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› But did not overcome these basic problems
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• We all wanted a DNA cutting enzyme (a nuclease) that:

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› Cuts at a sequence of our choosing

› Would work inside living cells

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 Enter targetted nucleases
Zinc finger nucleases and TALENs
• A new protein required for each site to be cut
• Proprietary technology

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• Expensive

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• Efficiency limited

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Walker-Daniels J, Mater. Methods, 2013
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 CRISPR/Cas9 (and variants)
Lives up to the hype!
• Open source (for research)
• Cheap

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• Highly efficient

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gRNA Cas9

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Clustered
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Regularly
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Interspaced
Short
Palidromic
Repeats

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Pennisi E, Science, 2013
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 Two ways to edit DNA with CRISPR/Cas9

gRNA/Cas9
Genome to be edited

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Homology-directed

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repair (HDR)

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Non-homologous
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end joining (NHEJ)

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 Why is it so easy?
• There are genetic constructs that make Cas9 and have
a site to add DNA for any gRNA you like
› https://www.addgene.org/CRISPR/

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• If this is put into cells, it just goes to work…

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Cas9

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FOI 240-1718 9 Pennisi E, Science, 2013

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 So very easy
• You can buy Cas9 protein and any gRNA you like
• Mix them in a tube
• ‘Transfect’ them into a cell

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› with or without a repair DNA

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• If there is no DNA, is it a GMO?

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$200
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Pennisi E, Science, 2013
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 The catch: Specificity and ‘off-target effects’

• Off-target effects are considered the main limitation

› Cutting at undesired locations

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› Partly addressed by combining two Cas9 ‘nickases’

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5’ 3’
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3’ 5’

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 Gene therapy can now include gene correction

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CRISPR/Cas9

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Repaired cells Diseased cells

Targetting the CRISPR/Cas9 is still a major hurdle for in vivo gene therapy

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‘CRISPR’ clinical trials on the US NIH database

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Gene drives

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 Gene drives

• Genes that are inherited at greater than Medelian rates

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SYNTHETIC GENE DRIVES IN AUSTRALIA:

IMPLICATIONS OF EMERGING TECHNOLOGIES
AUSTRALIAN ACADEMY OF SCIENCE MAY 2017

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 Normal (Mendelian) inheritance
• There are two copies of the genome
• One copy of each gene comes from each parent

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The gene will be in ¼ of next generation (assuming most are grey)

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Gene drives

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• A gene drive can cause a gene to be duplicated

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What does CRISPR have to do with gene drives?

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 Applications of gene drives
• Inhibiting / controlling insect vectors of disease
› Health benefits, especially for developing nations
• Pest animal control

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› Potential environmental benefit

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• Controlling pests of agriculture

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› Commercial benefit

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› Improved food security

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• Any release requires enourmous care and

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consideration
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• Public scrutiny required

› Very substantial dread

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 Strategic issues

• How does Australia ensure we are active participants

and not bystanders?

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• How will we decide when genome engineering is

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considered safe for human medicine?

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› What will be the quality benchmarks for certification?

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• How will we balance potential health benefits against

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environmental concern (e.g. gene drives)?
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• How will we ensure that policy and the public are
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informed by science?
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• How will we respond to international regulatory moves

(e.g. licencing or moratoria)

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