Promises and challenges of gene editing in the age of CRISPR. Neville Sanjana New York Genome Center & NYU Biology
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1 Promises and challenges of gene editing in the age of CRISPR Neville Sanjana New York Genome Center & NYU Biology
2 We have developed advanced tools to manipulate information stored in books or on computers
3 But, until recently, we have had few tools for easy manipulation of DNA DNA = The language of all living things?
4 Targeted modification of complex genomes Genome engineering takes advantage of natural gene repair to introduce novel genetic sequences. Jellyfish GFP mice Vancouver Aquarium Okabe et al. (1997)
5 Targeted double strand breaks enable genome editing in mammalian cells Knock-out Knock-in Rudin et al. 1989, Plessis et al (Haber lab) Rouet et al (Jasin lab) Bibikova et al. 2001, 2002, 2003 (Carroll lab)
6 How do we make precise cuts in DNA?
7 CRISPR: A tool for editing genomes Science (2013) Science (2015)
8 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
9 CRISPR-Cas9 is easier to target to multiple genomic loci Protein specifies DNA targeting RNA specifies DNA targeting Zinc finger nucleases CRISPR-Cas9 Kim et al. (1996) Transcription Activator-Like Effector Nucleases (TALENs) Boch et al. (2009), Mouscou et al. (2009), Christian et al. (2010), Miller et al. (2011) 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)
10 A brief history of CRISPR Hsu et al. Cell (2014)
11 1987: Repetitive DNA sequences in prokaryotes Hsu et al. Cell (2014) CRISPR = Clustered Regularly Interspaced Palindromic Repeats
12 2007: First evidence for CRISPR as an adaptive immune system Hsu et al. Cell (2014)
13 Insights from yogurt: An adaptive immune system in prokaryotes Mojica et al. (2005), Pourcel et al. (2005) Barrangou et al. (2007)
14 Nature (2016)
15 CRISPR has had a big impact in just a few years Food security Engineered food products Wheat Powdery mildew resistance Wang et al. (2014) Cancer immunotherapy Mushrooms Non-browning mushroom Yang et al. (2015) Infectious disease Engineered T cells More finely tuned to kill cancer Many groups Malaria resistance Self-copying gene drives in mosquitos Gantz et al. (2015)
16 Ethical issues concerning genetic engineering in humans Germline vs Somatic
17 Survey data from ~1600 US adults Two-thirds approved of both somatic and germline procedures for therapeutic purposes. A. Dane, Nature Biotech (2017)
18 Many diseases can be avoided via pre-implantation genetic diagnosis (PGD) already in the clinic
19 Precision medicine: Finding a needle in a haystack How can we efficiently identify which regions of the genome drive disease?
20 From targeting a single gene in the genome.
21 Deleting every gene in the genome in parallel
22 Harnessing CRISPR-Cas9 for genome engineering Targeted gene knockout or replacement 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)
23 Designing large libraries of guide RNAs guides on a single chip 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)
24 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)
25 Key idea: A pooled genetic screen can scale efficiently Each cell receives ONE genetic manipulation Measure enrichment or depletion after selection Science (2014)
26 Finding genes that drive resistance to cancer therapies Vemurafenib: FDA-approved drug for malignant melanoma Untreated Before vemu Remission 15 weeks vemu Recurrence 24 weeks vemu Mutation Wagle*, Emery* et al. (2011)
27 Vemurafenib pooled screen Science (2014)
28 After drug treatment there is high enrichment of a small group of sgrnas Science (2014)
29 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)
30 Raf inhibitor resistance mechanisms from validated GeCKO screen hits are in key cancer pathways Ub CUL3 NF1 Hollstein and Chicowski (2013) Corrall et al. (2003) MED12 NF2 TGFβ-R2 Huang et al. (2012) MET signaling Shrestha et al. (2012) mtorc1 & 2 James et al. (2009) James et al. (2012) hippo Murray et al. (2012) Lito et al. (2013) TADA1 TADA2B CCDC101 STAGA complex MYC Liu et al. (2008)
31 GeCKO screen targets are mutated in exome sequencing of vemurafenib-resistant tumors Ub CUL3 NF1 Hollstein and Chicowski (2013) Corrall et al. (2003) MED12 NF2 TGFβ-R2 Huang et al. (2012) MET signaling Shrestha et al. (2012) mtorc1 & 2 James et al. (2009) James et al. (2012) hippo Murray et al. (2012) TADA1 TADA2B CCDC101 Lito et al. (2013) STAGA complex MYC Liu et al. (2008) n = 31 patients Van Allen et al. (2014)
32 Thank you! 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
33 Legal notice 2017 Swiss Re. All rights reserved. You are not permitted to create any modifications or derivative works of this presentation or to use it for commercial or other public purposes without the prior written permission of Swiss Re. The information and opinions contained in the presentation are provided as at the date of the presentation and are subject to change without notice. Although the information used was taken from reliable sources, Swiss Re does not accept any responsibility for the accuracy or comprehensiveness of the details given. All liability for the accuracy and completeness thereof or for any damage or loss resulting from the use of the information contained in this presentation is expressly excluded. Under no circumstances shall Swiss Re or its Group companies be liable for any financial or consequential loss relating to this presentation. 33
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