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Applications of CRISPR-
Cas9 for disease research
Randall J. Platt, Ph.D.
Feng Zhang Lab,
Broad Institute of MIT and Harvard
Engineering the genome to
investigate disease mechanisms
Randall J Platt, PhD Broad | MIT | McGovern
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agcatcaagaagaacctgatcggagccctgctgttcgaca
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Patients
Disease
Genotype Phenotype
Therapy
Model systems
Targeted DNA breaks facilitate genome editing
Knock-out Knock-in
Targeted genome editing toolbox
• ZF
• TALE
• ZFN/TALEN
• CRISPR-Cas9
CRISPR: a microbial adaptive immune system
CRISPR-Cas9
• Guide RNA + Cas9
• 20 nucleotide target
• DNA double strand break
• Multiplexable
Jinek et al. Science 2012, Gausianas et al. PNAS 2012, Cong et al. Science 2013, Mali et al Science 2013.
Gene editing with Cas9
in vitro in cell lines Cong et al. Science 2013
Mali et al. Science 2013
Jinek et al. eLife 2013
Cho et al. Nat Biotechnol 2013
and others
single cell zygotes Hwang et al. Nat Biotechnol 2013
Wang et al. Cell 2013
Niu et al. Cell 2014
and others
Edit genes directly in vivo in adult mice
Cas9 sgRNA sgRNA
Cas9 mouse
Controlled Cas9 expression by Cre crosses
Neuronal subtype
specific expression
Ubiquitous expression
TH Cas9 TH/Cas9
PV Cas9 PV/Cas9
Zoom
Zoom
Platt et al. Cell 2014
Is Cas9 toxic in vivo?
LSL-Cas9 WT Cas9
Platt et al. Cell 2014
LSL-Cas9 WT
Cas9
Cas9-mediates efficient gene KO
Platt et al. Cell 2014
Cas9-mediates efficient gene KO
Platt et al. Cell 2014
Cas9-mediates efficient gene KO
Western blot
Western blot quant Indel analysis by NGS
NeuN
Platt et al. Cell 2014
NeuN Platt et al. Cell 2014
From monogenic to polygenic
Polygenic disease modeling
• Simultaneously model top three lung cancer alleles • P53, LKB1, KRAS
Platt et al. Cell 2014
Polygenic disease modeling
• Simultaneously model top three lung cancer alleles • P53, LKB1, KRAS
Saline AAV9-luciferase
Platt et al. Cell 2014
Polygenic disease modeling
• Simultaneously model top three lung cancer alleles • P53, LKB1, KRAS
sgLacZ Kras, p53, and Lkb1
(KPL)-targeted
Platt et al. Cell 2014
Polygenic disease modeling
• Simultaneously model top three lung cancer alleles • P53, LKB1, KRAS
sgLacZ Kras, p53, and Lkb1 (KPL)-targeted
Platt et al. Cell 2014
Genome engineering with CRISPR-Cas9 mice
Acknowledgements Feng Zhang
Ian Slaymaker
Naomi Habib
Lukasz Sweich
Hannah Kempton
Matthias Heidenreich
Michael Yim
Zhang lab
Phil Sharp
Sidi Chen
Guoping Feng
Yang Zhou
Robert Langer
Daniel Anderson
James Dahlman
Siddharth Jhunjhunwala
Aviv Regev
Oren Parnas
Marko Jovanovic
Nir Hacohen
Thomas Eisenhaure
Ramnik Xavier
Daniel Graham
Personal support: NSF GRFP, Afeyan, and Friends of McGovern Fellowships
Research support: NIH Pioneer and TR01 awards, NSF Waterman; the Keck, Damon Runyon,
Searle Scholars, Klingenstein, Vallee, Merkin, and Simons Foundations, and Bob Metcalfe
Resources:
Cas9 mice available through
The Jackson Laboratory
Cre-dependent - 024857
Constitutive - 024858
DNA plasmids available through Addgene
Questions?
rjplatt@mit.edu
Learn more
27
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Webinar Archives:
www.genscript.com/webinars.html
CRISPR Resource Center:
www.genscript.com/CRISPR.html
Thank you!
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Applications of CRISPR-Cas9 mice
Immune cell editing ex vivo
Vasculature genome editing by
nanoparticle-mediated delivery of sgRNA
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