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CRISPR: Opportunities and Challenges Webinar

Your Personal Research Assistant

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Webinar Host

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Medical Lead Scientist at PreScouter, Inc.

Ph.D. Biophysical Sciences from the University of Chicago

Charles Wright, Ph.D.

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What is CRISPR/Cas?

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): repeating palindromic sequences of

DNA in prokaryotes; unique spacer DNA sequences matching to foreign DNA are found between each repeat

Cas (CRISPR Associated protein): enzymes needed to identify and cut the targeted DNA sequence

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What is CRISPR/Cas?

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CRISPR/Cas: form of acquired immunity in prokaryotes that confers resistance to foreign genetic elements

CRISPR/Cas9: simplified, programmable version of CRISPR/Cas modified to edit genomes

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How the CRISPR/Cas9 system works

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sgRNA (single guide RNA): synthetic RNA molecule that contains the components needed to target the desired

genomic DNA sequence(s) and to complex with a Cas protein

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How the CRISPR/Cas9 system works

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Cas9: a Cas endonuclease protein that can cleave almost any DNA sequence complementary to its guide RNA

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How the CRISPR/Cas9 system works

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How the CRISPR/Cas9 system works

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials11

CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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CRISPR Timeline

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1987

2002

2005

2006

2007

2010

2012

2013

2014

2015

2016

2017

CRISPR repeats first observed in bacterial genomes

CRISPR elements and associated genes identified and named

CRISPR spacer identified as foreign DNA

CRISPR proposed to be a bacterial adaptive immune system

Discovery that CRISPR/Cas imparts resistance to specific phages

CRISPR/Cas identified as bacterial and archaeal immune system

CRISPR/Cas9 developed as gene editing tool

CRISPR/Cas9 used to edit targeted genes in both human and mouse cells

First use of CRISPR/Cas9 in plants

CRISPR/Cas9 used to cut HIV out of genome of infected humans cells

Monkeys with CRISPR-engineered targeted mutations are born

CRISPR/Cas9 used to develop virus-resistant tomato plants

CRISPR/Cas9 used to edit human embryos (but many off-target effects observed)

International moratorium proposed on making heritable changes to the human genome

USDA determines CRISPR/Cas9 edited crops will not be regulated as GMOs

First human trial to use CRISPR gene editing gets NIH approval

US patent office awards key CRISPR/Cas9 patents to the Broad Institute

National Academy of Sciences report outlines criteria to be met for germline editing clinical trials

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Potential Applications of CRISPR

Biomedical research:

we are creating new animal models

to understand disease and to aid in

drug discovery

Agriculture:

we can edit the genes of crops to

make them tastier, more nutritious,

pathogen-resistant, or more robust to

extreme environmental conditions

Gene drives:

instead of modifying just a single

organism, we could modify an entire

species

New antibiotics and antivirals:

we could use this system to eradicate

specific bacteria and viruses

New tools to stop genetic diseases:

we could edit the human genome to

eliminate genetic diseases like

Huntington’s disease, sickle cell

anemia, or cystic fibrosis

Designer humans:

we might one day edit the human

genome to eliminate disease or select

for desirable traits

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Your Personal Research Assistant

Technology Scouting

Market Analysis

Competitive Intelligence

Technology Evaluation

19

Technology Landscapes

Start up Interviews

Forecasts

Market Sizing

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3 Step Process

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STEP 1

Describe your questions Meet with your PreScouter-managed

Scholar team 2–3 times over 4–6 weeks

to review what they have found

STEP 2

Make smarter decisions,

based on the

Scholars’ report

STEP 3

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CRISPR Panelists

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John G. Doench, Ph.D.

Associate Director of the

Genetic Perturbation

Platform at the

Broad Institute

C.B. Gurumurthy,

Ph.D., Exec. MBA

Associate Professor at the

Department of

Developmental

Neuroscience, Munroe

Meyer Institute for Genetics

and Rehabilitation

Shuibing Chen, Ph.D.

Assistant Professor in the

Department of Surgery and

Biochemistry at Weill

Cornell Medical College

Questions

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What enhancements to the CRISPR/Cas9 system do

you think will be the most impactful?

1

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Of the current technical limitations facing the CRISPR technology,

which do you think are most important?

2

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What are the lessons we can learn from the obstacles faced by

CRISPR’s predecessors? In what ways is CRISPR unique from

these systems?

3

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What are the main technical challenges of applying CRISPR to

animal models? How will CRISPR-based development of

animal models allow us to create better disease models and

aid in drug discovery?

4

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5

How can CRISPR aid in research involving pluripotent stem cells?

What translational/therapeutic use cases do you foresee? What are

the hurdles to achieving these applications?

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CRISPR and Pluripotent Stem Cells (PSCs)

Neuron Blood Cells Cardiomyocytes β cells Liver Cells

Modified from Regenerative Medicine. August 2006

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Research Tools

• Create reporter line.

• Evaluate the biological function of certain genes or mutations in human development and human cells.

hPSCs definitive endoderm

pancreatic progenitor

glucose-responding cells

Isogenic hPSCs

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Therapeutic Application

Patient

Corrected

hPSCs

Reprogramming

Patient-specific

hPSCs

Directed

differentiation

Replacement

therapy

Patient

Corrected

tissue or organs

Isogenic

hPSCs Patient-specific

tissues or organs

Drug screening

Drug candidates for

precision therapy

Fo

ld C

ha

ng

e o

f IN

S+ C

ell

De

ath

Ra

te

#of INS+ cells

0.01

0.10

1.00

10.00

-100 100 300 500 700 900 1100 1300 1500

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What are the greatest challenges (technical, regulatory, or ethical) to

broader applications of CRISPR within biomedical research, drug

discovery, agricultural enhancements, or therapeutic applications?

6

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What applications of CRISPR are you most excited to see in the next

few years? What about the next 10–20 years?

7

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Future Direction of CRISPR in Pluripotent Stem Cells

1. Gene/Mutation Specific Drugs

Patient-specific

tissues or organs

Drug screening

Drug candidates for

precision therapy

Fo

ld C

ha

ng

e o

f IN

S+ C

ell

De

ath

Ra

te

#of INS+ cells

0.01

0.10

1.00

10.00

-100 100 300 500 700 900 1100 1300 1500

2. Isogenic iPSC-based Genome-Wide Association Study

Genome-Wide Association Study Gene-Environment Interaction

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