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

gacaagaagtacagcatcggcctggacatcggcaccaact

ctgtgggctgggccgtgatcaccgacgagtacaaggtgccc

agcaagaaattcaaggtgctgggcaacaccgaccggcac

agcatcaagaagaacctgatcggagccctgctgttcgaca

gcggcgaaacagccgaggccacccggctgaagagaacc

gccagaagaagatacaccagacggaagaaccggatctg

ctatctgcaagagatcttcagcaacgagatggccaaggtgg

acgacagcttcttccacagactggaagagtccttcctggtgg

aagaggataagaagcacgagcggcaccccatcttcggca

acatcgtggacgaggtggcctaccacgagaagtaccccac

catctaccacctgagaaagaaactggtggacagcaccgac

aaggccgacctgcggctgatctatctggccctggcccacat

gatcaagttccggggccacttcctgatcgagggcgacctga

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

www.genscript.com/CRISPR.html

Learn more

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CRISPR Resource Center:

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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