Using CRISPR to Study

Developmental Disorders

Bryan Luikart, PhD

Associate Professor of Molecular and Systems Biology

DNA Sequencing

The late 1980's.

An international team of scientists began the project to map the human genome.

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.

Nature | News Feature 19 March 2014

2008 – maybe ¼ of ASD was genetic in origin

(Abrahams and Geschwind, 2008; Kelleher and Bear, 2008)

Gaugler et

al. 2014

2014 – Over half of cases are genetic in origin

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

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s

ASD Candidate Gene

SFARI Database Genes (Cat S, 1, 2 and 3)

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?

Biological Warfare Between Bacteria and Viruses

CRISPR/Cas9 Cuts DNA – that’s all

Scientific American 311, 42 - 46 (2014) Published online: 18 November 2014

doi:10.1038/scientificamerican1214-42

Classic DNA repair mechanisms control what you can manipulate

after you cut the DNA

How to get protein - Cas9

RNA – sgRNA

& DNA – ssoDNA

.. into a cell

Transfection

Infection

Injection

Transfection

Variations of ripping holes in cells

CaPO4, liposomes, electroporation

Used on cells in vitro

Transient (but CRISPR is permanent)

rabies

Different Viral Vector Systems – advantages and disadvantages

Cell death

6 days

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

Pten acts as a negative regulator of the mTOR pathway

Laplante and Sabatini, J Cell Sci. (2009)

The Enlarged Brain of the Conditional Knockout is due to Neuronal Hypertrophy

Is this Relevant for Patients with PTEN Mutations?

Williams et al, J. Neurosci 2015

Does the Pten KO Phenotype Model

Patients With Mutations?

Cre-lox Gives Strong Phenotype but you need mice

Williams, Fricano et al., Scientific Reports 2016

shRNA Results in Weak Phenotype

Williams, Fricano et al., Scientific Reports 2016

CRISPR Lets You Model Mutations found in Pateints

Williams, Fricano et al., Scientific Reports 2016

A Single sgRNA Yields a Mixture of “wild-type” and “KO”

cells

Williams, Fricano et al., Scientific Reports 2016

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

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

Transgenic Technology – Zygote Injection

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

Knockout/Knockin Technology – ES cells

Knockout/Knockin Technology – transfect ES cells

Knockout/Knockin Technology – homologous recombination

Knockout/Knockin Technology – Blastocyst injection

Knockout/Knockin Technology – Chimeric Mice

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

CRISPR Gives you KO in the time it takes to make a transgenic

KATNAL2 – Katanin Catalytic Subunit A-Like 2

Katanin and Spastin are Microtubule Severing Enzymes

Williams et al., Scientific Reports 2016

Katnal2 KO Neurons Have Decreased Dendritic Arborization and

Increased Dendritic Spine Density

CRISPR Katnal2 KO Mice

Set 1 – NHEJ KO

15 pups - 4 INDELS

Set 2 – HDR point mutation

8 pups – 4 INDELS,

1 HDR

Mouse Models May Lead to New Treatments

Mouse Baby Nova Lise Luikart

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