using crispr to study developmental disorders · katanin and spastin are microtubule severing...
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Using CRISPR to Study
Developmental Disorders
Bryan Luikart, PhD
Associate Professor of Molecular and Systems Biology
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DNA Sequencing
The late 1980's.
An international team of scientists began the project to map the human genome.
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Human Genome Project
Imagine a world in which we will be able to treat diseases by altering our very genes‚ giving us new ones if ours are non-functional, changing bad genes for good ones. For the first time in our existence, we are closer to understanding just what we are. We now have the tools to make the whole world better through science ‚ the science of the human genome.
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Nature | News Feature 19 March 2014
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2008 – maybe ¼ of ASD was genetic in origin
(Abrahams and Geschwind, 2008; Kelleher and Bear, 2008)
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Gaugler et
al. 2014
2014 – Over half of cases are genetic in origin
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ada
adnp adsl ahi1 aldh5a1 ank2 ankrd11 ankrd11 anxa1 aph1a arid1b arx ash1l astn2 asxl3 atp10a atp2b2 auts2
avpr1a
bckdk bcl11a bps13b cacna1c cacna1d cacna1h cacnb2 cc2d1a cdc42bpb cdh7 cdkl5 cep41 chd2 chd2 chd8
chrna7
cntn4 cntnap2 cntnap4
ctcf
ctnnb1
cttnbp2 cul3 deaf1 dhcr7 dip2a
disc1
dmd
dmpk
dscam dyrk1a efr3a ehmt1 ehmt1 elp4 etfb
fmr1
foxp2 gabrb3 gigyf1 gphn grik2 grin2b grip1 hdac4 hepacam hmgn1 hoxa1 itgb3
katnal2 kcnj10 kdm5b kdm6b kmt2a kmt2c kmt2e lamb1 lmx1b macrod2 magel2 mbd5 mbd5
mecp2
med13l msnp1as myo9b myt1l naa15 nckap1
nf1
nipbl nlgn3 nlgn4x
nr3c2
nrxn1 nrxn3 nsd1 ntng1 ophn1 oxtr pax5
pcdh19 phf2 pogz
pon1
prkcb ptchd1
pten
rai1 rbfox1 reln rims1 scn1a scn2a sema5a setbp1 setd5 setd5 shank1 shank2 shank3 slc38a10
slc6a3
slc9a6 spast suv420h1 syne1 syngap1 tbr1
tcf7l2
tmlhe tnrc6b
trio
trip12 trpc6 tsc1 tsc2
ube3a upf3b vil1 wac wdfy3 zbtb20 zmynd11 0
5000
10000
15000
20000
25000
0 20 40 60 80 100 120 140
Pu
bm
ed C
itati
on
s
ASD Candidate Gene
SFARI Database Genes (Cat S, 1, 2 and 3)
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CRISPRs, Clustered Regularly
Interspaced Short Panlindromic Repeat
sequences are part of an adaptive immune system used by bacteria to
target foreign DNA, eg. phage virus or plasmid. In its most
basic form, the system is composed of RNA transcribed from
CRISPR regions and multiple CRISPR associated (Cas)
proteins. The Cas proteins bind to the CRISPR RNAs to effect
processing of RNA and nuclease attack of foreign DNA.
From Chen and LoTurco ATMN 2014
CRISPR – What does it stand for?
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Biological Warfare Between Bacteria and Viruses
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CRISPR/Cas9 Cuts DNA – that’s all
Scientific American 311, 42 - 46 (2014) Published online: 18 November 2014
doi:10.1038/scientificamerican1214-42
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Classic DNA repair mechanisms control what you can manipulate
after you cut the DNA
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How to get protein - Cas9
RNA – sgRNA
& DNA – ssoDNA
.. into a cell
Transfection
Infection
Injection
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Transfection
Variations of ripping holes in cells
CaPO4, liposomes, electroporation
Used on cells in vitro
Transient (but CRISPR is permanent)
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rabies
Different Viral Vector Systems – advantages and disadvantages
Cell death
6 days
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Butler et al 2005
Buxbaum et al 2007
Herman et al 2007
Stein et al 2010
McBride et al 2010
etc….
Increased Brain Size Decreased Social Interaction
Inappropriate Response Learning Deficits
Kwon CH*, Luikart BW*, Powell CM*, Zhou J, Matheny SA, Zhang W, Li Y, Baker SJ, Parada LF. (2006) Neuron 50:377-388
Pten Regulates Neuronal Arborization and Social Interaction in Mice
increased brain size decreased social interaction
inappropriate response Intellectual disability
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Pten acts as a negative regulator of the mTOR pathway
Laplante and Sabatini, J Cell Sci. (2009)
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The Enlarged Brain of the Conditional Knockout is due to Neuronal Hypertrophy
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Is this Relevant for Patients with PTEN Mutations?
Williams et al, J. Neurosci 2015
Does the Pten KO Phenotype Model
Patients With Mutations?
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Cre-lox Gives Strong Phenotype but you need mice
Williams, Fricano et al., Scientific Reports 2016
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shRNA Results in Weak Phenotype
Williams, Fricano et al., Scientific Reports 2016
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CRISPR Lets You Model Mutations found in Pateints
Williams, Fricano et al., Scientific Reports 2016
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A Single sgRNA Yields a Mixture of “wild-type” and “KO”
cells
Williams, Fricano et al., Scientific Reports 2016
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Transgenics – if you want to “CRISPR” every cell in the mouse
Yoshiko Nakagawa et al. Biology Open 2016;bio.019349
© 2016. Published by The Company of Biologists Ltd
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Figure 4 - Harvesting of zygotes.
From the following article Pronuclear injection for the production of transgenic mice
Lars M Ittner & Jürgen Götz
Nature Protocols 2, 1206 - 1215 (2007)
Published online: 10 May 2007
doi:10.1038/nprot.2007.145
Transgenic Technology - harvest the zygotes
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Transgenic Technology – Zygote Injection
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Figure 7 - Reimplantation of injected zygotes. Pronuclear injection for the production of transgenic mice
Lars M Ittner & Jürgen Götz
Nature Protocols 2, 1206 - 1215 (2007) Published online: 10 May 2007
doi:10.1038/nprot.2007.145
Transgenic Technology - implant zygote into pseudopregnant female
- Induce pseudopregnancy by
mating females in estrous
with vesectomized males
- Inject zygotes into the
oviduct
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Knockout/Knockin Technology – ES cells
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Knockout/Knockin Technology – transfect ES cells
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Knockout/Knockin Technology – homologous recombination
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Knockout/Knockin Technology – Blastocyst injection
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Knockout/Knockin Technology – Chimeric Mice
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Transgenic Technology Knockout Technology
-Faster
-Does not target specific spot in
genome
-Traditional vs BAC
-slow growing cells and
breeding
-HR is super inefficient
-does target specific
locus
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CRISPR Gives you KO in the time it takes to make a transgenic
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KATNAL2 – Katanin Catalytic Subunit A-Like 2
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Katanin and Spastin are Microtubule Severing Enzymes
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Williams et al., Scientific Reports 2016
Katnal2 KO Neurons Have Decreased Dendritic Arborization and
Increased Dendritic Spine Density
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CRISPR Katnal2 KO Mice
Set 1 – NHEJ KO
15 pups - 4 INDELS
Set 2 – HDR point mutation
8 pups – 4 INDELS,
1 HDR
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Mouse Models May Lead to New Treatments
Mouse Baby Nova Lise Luikart
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Luikart Lab
Stephanie Getz
Michael Williams
Jessie Poquerusse
Patrick Skelton
Julia Salinaro
Paul Frazel
Meijie Li
Katie Fricano-Kugler
Collaborators – Dartmouth-
Dave Bucci
Kyle Smith
Allan Gulledge
Hermes Yeh
Cincinnati Children’s-
Christina Gross
UCSD-
Chitra Mandyam