Promises and challenges of gene editing in

the age of CRISPR

Neville Sanjana New York Genome Center & NYU Biology

We have developed advanced tools to manipulate

information stored in books or on computers

But, until recently, we have had few tools for easy

manipulation of DNA

DNA = The language of all living things

?

• Genome engineering takes advantage of natural gene repair

to introduce novel genetic sequences.

GFP mice

Okabe et al. (1997)

Targeted modification of complex genomes

Jellyfish

Vancouver Aquarium

Targeted double strand breaks enable genome

editing in mammalian cells

Rudin et al. 1989, Plessis et al. 1992 (Haber lab)

Rouet et al. 1994 (Jasin lab)

Bibikova et al. 2001, 2002, 2003 (Carroll lab)

Knock-out Knock-in

How do we make precise cuts in DNA?

CRISPR: A tool for editing genomes

Science (2015)

Science (2013)

The Model T of writing DNA “The Model T wasn’t the

first car, but it changed

the way we drive, work,

and live. CRISPR has

made a difficult process

cheap and reliable.”— Hank Greely

CRISPR-Cas9 is easier to target to multiple genomic loci

Zinc finger nucleases

Transcription Activator-Like Effector

Nucleases (TALENs)

CRISPR-Cas9

Boch et al. (2009), Mouscou et al. (2009),

Christian et al. (2010), Miller et al. (2011)

Kim et al. (1996)

Protein specifies DNA targeting RNA specifies DNA targeting

Garneau et al. (2010), Sapranauskas et al. (2011),

Jinek et al. (2012), Gasiunas et al. (2012)

Cong*, Ran* et al. (2013), Mali*, Yang* et al. (2013)

Hsu et al. Cell (2014)

A brief history of CRISPR

Hsu et al. Cell (2014)

1987: Repetitive DNA sequences in prokaryotes

CRISPR =Clustered Regularly Interspaced Palindromic Repeats

Hsu et al. Cell (2014)

2007: First evidence for CRISPR as an adaptive

immune system

Mojica et al. (2005), Pourcel et al. (2005)

Barrangou et al. (2007)

Insights from yogurt:

An adaptive immune system in prokaryotes

Nature (2016)

CRISPR has had a big impact in just a few years

Wheat | Powdery mildew

resistance Wang et al. (2014)

Mushrooms | Non-browning

mushroom Yang et al. (2015)

Engineered T cells | More finely

tuned to kill cancer Many groups

Malaria resistance | Self-copying gene

drives in mosquitos Gantz et al. (2015)

Food security Engineered food products

Cancer immunotherapy Infectious disease

Ethical issues concerning genetic engineering in

humans

Germline vs Somatic

Survey data from ~1600 US adults

A. Dane,

Nature Biotech (2017)

“Two-thirds

approved of both

somatic and

germline

procedures for

therapeutic

purposes.”

Many diseases can be avoided via pre-implantation

genetic diagnosis (PGD) — already in the clinic

Precision medicine: Finding a needle in a haystack

How can we efficiently identify which regions of the genome drive disease?

From targeting a single gene in the genome….

Deleting every gene in the genome in parallel

Targeted gene knockout or replacement

Harnessing CRISPR-Cas9 for genome engineering

Garneau et al. (2010), Sapranauskas et al. (2011),

Jinek et al. (2012), Gasiunas et al. (2012)

Cong*, Ran* et al. (2013), Mali*, Yang* et al. (2013)

Designing large libraries of guide RNAs

Garneau et al. (2010), Sapranauskas et al. (2011),

Jinek et al. (2012), Gasiunas et al. (2012)

Cong*, Ran* et al. (2013), Mali*, Yang* et al. (2013)

104 - 106 guides

on a single chip

Genome-scale CRISPR Knock-Out (GeCKO) screening:

Targeted knock-out of all genes in the human genome

sgRNA library

Key idea: Target Cas9 to different genes and take advantage of

NHEJ-mediated error-prone repair to create loss-of-function.

Science (2014)

Key idea: A pooled genetic screen can scale efficiently

Each cell receives

ONE genetic manipulation

Measure enrichment or

depletion after selection

Science (2014)

Finding genes that drive resistance to cancer therapies

Vemurafenib: FDA-approved drug for malignant melanoma

Before vemu 15 weeks vemu

Wagle*, Emery* et al. (2011)

Untreated Remission

24 weeks vemu

Recurrence

Mutation

Vemurafenib pooled screen

Science (2014)

After drug treatment there is high enrichment

of a small group of sgRNAs

Science (2014)

Identification of true positive hits based on consistency

of unique sgRNAs targeting the same gene

Two GeCKO screen hits have recently been shown to confer vemurafenib resistance:

• MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGFb Receptor

Signaling. Huang et al. Cell (2012).

• A Genome-Scale RNA Interference Screen Implicates NF1 Loss in Resistance to RAF Inhibition.

Whittaker et al. Cancer Discovery (2013). Science (2014)

Raf inhibitor resistance mechanisms from validated

GeCKO screen hits are in key cancer pathways

Lito et al. (2013)

NF1

TGFβ-R2

MED12

Huang et al. (2012)

NF2

MET

signalingShrestha et al.

(2012)

mTORC1

& 2James et al.

(2009)

James et al.

(2012)

hippoMurray et al. (2012)

CUL3

TADA1

TADA2BCCDC101

STAGA complex

MYCLiu et al. (2008)

Hollstein and

Chicowski (2013)

Corrall et al. (2003)

Ub

GeCKO screen targets are mutated in exome

sequencing of vemurafenib-resistant tumors

Lito et al. (2013)

NF1

TGFβ-R2

MED12

Huang et al. (2012)

NF2

MET

signalingShrestha et al.

(2012)

mTORC1

& 2James et al.

(2009)

James et al.

(2012)

hippoMurray et al. (2012)

CUL3

TADA1

TADA2BCCDC101

STAGA complex

MYCLiu et al. (2008)

Hollstein and

Chicowski (2013)

Corrall et al. (2003)

Ub

n = 31 patients

Van Allen et al. (2014)

NYGC & NYU

Congyi Lu

Cathy Guo

Neil Bapodra

Meer Mustafa

Poonam Chitale

Antonino Montalbano

Neil Jethani

Bianca Diaz

Collaborators

Jason Wright

Xi Shi

Ophir Shalem

Kaijie Zheng

Jen Pan

Steve Hyman

Feng Zhang

Sidi Chen

Phil Sharp

Shashank Patel

Nick Restifo

Thank you!

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