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Transcript of Promises and challenges of gene editing in the age of ...a26e84be-b83a-43bb-9b85...TADA1 TADA2B...
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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