CRISPR: what it is, and why it is having a profound impact on human health

November 16, 2016
CRISPR: what it is, and why it
is having a profound impact on
human health
A Pistoia Alliance Debates Webinar
Chaired by Alvis Brazma – EMBL-EBI

This webinar is being recorded

Poll Question 1: How would you rate your
personal knowledge of CRISPR?
A. I’m an expert
B. I have used CRISPR
C. I’ve heard of it
D. I know next to nothing about it

©PistoiaAlliance
The Panel
4
Patrick Harrison, Senior Lecturer Physiology, University College of Cork
The focus of Dr Harrison’s lab is the development gene editing for treatment of rare diseases. His early work in this field
pioneered the use of ZFNs and CRISPR to successfully repair the most common CF-causing mutation, F508del, in cell
culture. The current focus extends the work to correct CF mutations of the deep intron theratype in primary cells, stem
cells and animal models. Dr. Harrison is a principal investigator in the CF Trust’s Gene Editing Strategic Research Centre,
and has additional grant funding from the CF Foundation (USA), with collaborations across Europe, USA and New Zealand.
He is also using CRISPR editing to model skin disorders such as atopic dermatitis and Epidermolysis Bullosa.
Alvis Brazma, Senior Team Leader of Functional Genomics, EMBL-EBI
Dr Alvis Brazma is a Senior Scientist at the European Molecular Biology Laboratory (EMBL) and leading a group on gene
expression at the European Bioinformatics Institute (EMBL-EBI). He studied mathematics at the University of Latvia, Riga,
before obtaining his PhD in computer science from the Moscow State University. In 1997 he joined the EMBL and in 1999
was among the first scientists to use microarray data to study gene regulation. In 1999 he founded the Microarray Gene
Expression Data society and started a microarray database ArrayExpress. Now he is in charge of several major EBI
resources, including ArrayExpress, Expression Atlas, and BioStudies database. He is co-leading the working group on data
integration for Pan-cancer Whole Genome project of the International Cancer Genome Consortium.
Mike Ollmann, Principal Scientist, Amgen
Dr. Ollmann’s work focuses on use of somatic cell genetics techniques, particularly CRISPR/Cas9 and RNAi, for early stage
drug discovery. Dr. Ollmann obtained his Ph.D. in Genetics with Dr. Greg Barsh at Stanford University, prior to joining
Exelixis Inc., where his work included early use of RNAi in Drosophila and mammalian cells for target identification and
validation. He joined Amgen in 2011 and is a Principal Scientist in the Genome Analysis Unit based in South San Francisco.
Anna Middleton, Head of Society & Ethics Research Wellcome Genome
Campus
November 16, 2016 CRISPR
Dr Anna Middleton is a social scientist, continually asking ‘how are people responding to genomics?’ Her PhD is in
psychology and she is also a trained genetic counsellor, having worked in the NHS for 10 years with patients exploring the
impact of genetics on themselves and their families. She runs the Society and Ethics Research Group at the Wellcome
Genome Campus in Cambridge and delivers research that explore the social and ethical impact of new genomic
technologies.

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Agenda
5
• CRISPR – Cas9 Gene Editing(PH)
• Accelerating science (MO)
• The informatics challenge (AB)
• Cystic Fibrosis - the case for gene-editing (PH)
• Ethics and CRISPR (slides from AM; presented
by PH)
5November 16, 2016 CRISPR

CRISPR – Cas9 Gene Editing
Why How What
Patrick Harrison, PhD – University College Cork, Ireland

Val His Leu Thr Pro Glu Glu Lys Ser Asp
November 16, 2016 CRISPR 7

Val His Leu Thr Pro Val Glu Lys Ser Asp
Target
DNA

Thr Pro Glu Glu Lys
Val His Leu Thr Pro Val Glu Lys Ser Asp
Target
DNA
Donor
DNA

Target
DNA
Donor
DNA
Clustered Regularly Interspaced Short Palindromic Repeats
Cas9
guide RNA

Target
DNA
Donor
DNA
Cas9
guide RNA
Cut Resect Incorporate Seal Precision Repair

Target
DNA
Donor
DNA
Cas9
guide RNA

Thr Pro Glu Glu Lys
Val His Leu Thr Pro Glu Glu Lys Ser Asp
HDR
Homology-directed repair

Target
DNA
Cas9
guide RNA

Val His Leu Thr Pro Gly Glu Val STOP
NHEJ
Non-homologous
end joining
In-frame STOP codon
= gene knock-out
November 16, 2016 22CRISPR

Cas9
(2013)
gRNA
Why has Cas9/gRNA surpassed ZFNs and TALENs?
ZFNs
(1996)
TALENs
(2011)
Standing on the
shoulders of giants
November 16, 2016 23CRISPR

Cas9/guideRNA
Design and synthesis (2013)

||||||||||||||||||||
5’ GUCACCUCCAAUCAGUAGGG 3'
gRNA
Cas9/guideRNA

||||||||||||||||||||
5’ GUCACCUCCAAUCAGUAGGGGUUUUAGAGCUAG
.|||||. ||||
GUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAUA
|||| GAA
AAAGUGGCACCGA
.|||||||G
3’ UUUUUUCGUGGCU
A
A
A
A
gRNA
Cas9/guideRNA

||||||||||||||||||||
5’ GUCACCUCCAAUCAGUAGGGGUUUUAGAGCUAG
.|||||. ||||
GUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAUA
|||| GAA
AAAGUGGCACCGA
.|||||||G
3’ UUUUUUCGUGGCU
A
A
A
A
Cas9
DSB
gRNA
Cas9/guideRNA

Double Stranded Break
Two options
NHEJ
• High efficiency KO
• All cell types
• KO in any animal
• Clinical Editing
HDR
• Low efficiency Precision
Repair
• Dividing cells only
• Multiple corrections in vivo

GFP
transgenic
rat
~40% Knock Outs
CRISPR 2016
Mice
Rats
Cows
Sheep
Rabbits
Monkeys
Zebrafish
Human Embryos
(14-day limit)
Transfer KO eggs
to surrogate
Pronuclei
Germline Editing
Inject Nuclease into Fertilised Eggs
Yang 2009

F.IX gene
variant
1 2 3 4
Pre-clinical in vivo editing – haemophilia B
i.v. inject nuclease and donor
Li 2011

F.IX gene
variant
1 2 3 4
Li 2011

F.IX gene
variant
Super-exon
Donor
1 2 3 4
2 3 4
F.IX gene
wild-type
1 2 3 42 3 4
Li 2011

Gene Editing
= Permanent
cDNA addition
= Transient
Li 2011

Gene Editing
= Permanent
cDNA addition
= Transient
Li 2011
Li 2011

100 to 1,000-fold
reduction in viral
load
Control
Tebas 2014
Patient #1
ZFNs delete CCR5

Acute Lymphoid Leukaemia
Modified Patient T cells
CAR targets CD19
TALENs delete TCR
(avoids rejection)
2015 Patient #2
TALENs enable CAR-T cells

CRISPR – Cas9 Gene Editing
Interim Summary
• Cas9/gRNA creates DNA breaks
• HDR – precision repair @ low efficiency
• NHEJ – targeted deletions @ high efficiency
• Gene-edited cells already used in patients
• CRISPR clinical trials – 2017/18?

Poll Question 2: Does your company have
an active CRISPR research/informatics
effort underway?
A. Actively using CRISPR
B. Exploring use of CRISPR
C. Not currently using CRISPR
D. I don’t know

Accelerating science
Mike Ollmann - Amgen

©PistoiaAlliance
Drug Discovery at Amgen
Target discovery & validation driven by human genetics

©PistoiaAlliance
Gene Knockout & Editing by CRISPR/Cas9
Figure from ThermoFisher

©PistoiaAlliance
Rapid Gene Knockout by Delivery of
Cas9/sgRNA Ribonucleoprotein complex
Liang et al, J Biotech, 2015
102 103 104 105
200
150
100
50
Flow cytometry to quantify target
protein levels 4 days after
electroporation of Cas9/sgRNA complex
Control cells - no sgRNA
Cells+Cas9/target sgRNA
Target protein expression levelCellcount

©PistoiaAlliance
Rapid Gene Knockout by Delivery of
Cas9/sgRNA Ribonucleoprotein complex
Not-so-rapid clonal isolation
of edited cells
Ran et al, Nature Protocols, 2013
Liang et al, J Biotech, 2015

©PistoiaAlliance
Pooled Lentiviral sgRNA Libraries for
Genome-scale Gene Knockout Screening
Figure adapted from Hartenian
& Doench, FEBS, 2015

©PistoiaAlliance
Beyond Gene Knockout: Alternative
Cas9-mediated Screening Methods
Sanjana, Analytical Biochem., 2016

Poll Question 3: Which is more important to
your research?
A. Precision editing by homology-directed
recombination (HDR)
B. Targeted knock-out/deletion by non-
homologous end-joining (NHEJ)
C. Don’t use CRISPR

The informatics challenge
From predicting the target sites to assessing the effects
Alvis Brazma – EMBL-EBI

©PistoiaAlliance
Why bioinformatics?
• Doing things in silico saves time and resources
• Some examples of what can be done
– Finding the target – a gene or locus of interest in the
genome
– Finding where to cut the genome near the target

©PistoiaAlliance
Cas9 nuclease is guided to the genome
position by 20 nt short guide RNA (sgRNA)
sequence
From Sander & Joung, Nature Biotech. 32, 247
The guide sequence

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Selecting the sgRNA sequence
Genome
Gene of interest
20 nt sequence
Most similar
sequence off
target
Target sequence
- perfect match
Most similar
sequence off
target
TCC

©PistoiaAlliance
Selecting the sgRNA sequence
Genome
Gene of interest
20 nt sequence
Most similar
sequence off
target
Most similar
sequence off
target
Target sequence
- perfect match
TCC TCC

©PistoiaAlliance
Annotated CRISPR/Cas9 target sites in
Ensembl genome browser at European
Bioinformatics Institute
Anna Farne, Mark Thomas, David Parry-Smith
November 16, 2016 CRISPR 54November 16, 2016 CRISPR 54

©PistoiaAlliance
What can bioinformatics do for CRISPR?
• Tools for predicting
and assessing the
short guide RNA
(sgRNA)
– Purely sequence
based methods
– Methods utilizing the
growing experimental
evidence
• Collecting the
successes and failures
of sgRNA sequences
• Using machine learning
to interpolate these
• Tools for assessing
and interpreting the
editing results
– Using and analyzing
direct effects based on
RNA sequencing
– Assessing further
downstream effects,
such as systematic
gene knockouts in cell
lines

©PistoiaAlliance
CRISPR activities at EMBL-EBI in
collaboration with the Wellcome Trust
Sanger Institute
• Computational annotation of sgRNA binding
sites
• (Planned) Curated database of experimental
results
– Experimentally validated sgRNA binding sites
– Knockout screen results
• Gene essentiality in various cell lines

Cystic Fibrosis
The Case for Gene Editing
Patrick Harrison, Ph.D. – University College Cork, Ireland

Cl-
Cl-
HCO3
-
HCO3
-
Airway hydrationNeutral pH
Normal Lung
CFTR anion channel
Cilia beating
H2O H2O

Cilia beating
CF Lung
Mutations in CFTR gene
collapsed
Cl-
Cl-
HCO3
-
HCO3
-
H2O H2O
Cilia collapsed

Class III
No conductance
Class II
Reduced Trafficking
Class I
No protein
CF – Personalised Medicine
4,000 people 92,000 people 4,000 people

Absolutechangein%
ofpredictedFEV1
Ramsay 2011
Nick Talbot
Class III
No conductance
4,000 people

Absolutechangein%
ofpredictedFEV1
.
Wainwright 2015
Orkambi (dose A)
Orkambi (dose B)
Placebo
BA
Class II
Reduced Trafficking
92,000 people

Absolutechangein%
ofpredictedFEV1
cDNA
Placebo
Alton 2015
Multi-dose
CFTR cDNA is
safe
Class I
No protein
4,000 people

64
Genome F508del
5'
3'
3’
5'
Donor CTT
3'
5'
5'
3'
CTT
GAA
---
---
Lee 2012

65
Genome F508del
5'
3'
3’
5'
Donor CTT
3'
5'
5'
3'
CTT
GAA
---
---
Lee 2012

66
Genome F508del
5'
3'
3’
5'
NGG
Donor CTT
3'
5'
5'
3'
CTT
GAA
---
---
Schwank 2013

67
5'
3'
3’
5'
3'
5'
5'
3'
CTT
GAA
Genome F508del
Donor CTT

68
5'
3'
3’
5'
3'
5'
5'
3' GAA
CTT
Genome F508del
Donor CTT

69
5'
3'
3’
5'
3'
5'
5'
3' GAA
CTT
Genome F508del
Donor CTT

70
5'
3'
3’
5'
3'
5'
5'
3' GAA
CTT
CTT
Genome F508del
Donor CTT

71
5'
3'
3’
5'
3'
5'
5'
3' GAA
CTT
CTT
GAA
CTT
Genome F508del
Donor CTT

72
Editing restores function
Stem Cell Organoids
WT F508del F508del edited
November 16,
2016
CRISPR
Schwank 2013
72

73
Stem Cell Organoids
November 16,
2016
CRISPR
Schwank 2013
73

74
Stem Cell Organoids
November 16,
2016
CRISPR
Schwank 2013
74

75
Schwank 2013
Stem Cell Organoids
November 16,
2016
CRISPR
75

76
HDR
restores
CFTR
<1%
Select &
enrich
NHEJ
>50%
KO
How can
KO fix CF?
76

77
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
CRISPR KO – 40% of Class I (and IV) mutations
Deep intron Mutations
3272 -26A>G
(n = 463)
3849 +10kb C>T
(n = 1,143)
* * *
1811+1.6kb A>G
(n = 71)
77

78
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
3849 +10kb C>T
(n = 1,143)
*
WT GTEx 22 AG GC AG Ex 23

79
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
3849 +10kb C>T
(n = 1,143)
*
WT GTEx 22 AG GT AG Ex 23
79

80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
3849 +10kb C>T
(n = 1,143)
*
WT GTEx 22 AG GT AG Ex 23pseudo
Exon
TAA
80
CRISPRNovember 16, 2016

81
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
3849 +10kb C>T
(n = 1,143)
*
GWT GTEx 22 GT AG Ex 23pseudo
Exon
TAA
Delete 25 to 150 bp
3849 +10kb C>T
(n = 1,143)
A
81

82
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 26 2724 25
3’5'
WT GTEx 22 AG Ex 23
82

83
November 16,
2016
CRISPR
One for All
CFTR super-exons
83

84
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
84

85
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
Bednarski 2016
CFTR Exons 11-27
85

86
CFTR Exons 11-27
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
Bednarski 2016
86

87
CFTR Exons 11-27
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
3’5'
Bednarski 2016
87

88
CFTR Exons 11-27
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
5'
CFTR Exons 11-27
CFTR Exons 11-27
3’
Bednarski 2016
88

89
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 2722 24 25
5' CFTR Exons 11-27
CFTR Exons 11-27
CFTR Exons 11-27
AAAA
3’
Unedited
Super-exon
Bednarski 2016
89

Cystic Fibrosis – Cas9 Gene Editing
Interim Summary
• HDR – precise but inefficient
• NHEJ – efficient but only 2% of individuals
• HDR superexon – all mutations but inefficient
• NHEJ superexon – TBC

Dr Anna Middleton
Head of Society and Ethics Research
Wellcome Genome Campus
Cambridge, UK

The most discussed ethics…
• The most controversial aspect of CRISPR is the
potential use in editing gametes or embryos
• It is illegal to edit a human embryo with the aim of
implanting it to achieve a pregnancy
• However, it is acceptable (e.g. in the UK, under
license) to do research using CRISPR on embryos up
to 14 days of age

Public Debate pivotal
• Public debate about ‘designer babies’, eugenics and
the ‘slippery slope’ in the application of genetic
technology has been happening for the last 40 years
• However, now is the time to consider, what is socially
acceptable in terms of research on embryos
• If parents consent for research to happen on their
discarded ’IVF’ embryos (that will never result in a
pregnancy), does this mean it is socially acceptable to
do?

The Debate so far…
• Should all research on embryos be banned?
• We live in a society where research on embryos up to
the 14 day point is acceptable (and is being done)
• CRISPR research should form part of this picture
• If there is a moratorium on editing embryos in a
research setting, this will push the research
underground and out of public scrutiny
• Research needs to be publicly funded on editing, in
order to maintain safe regulation

Help the debate…
• There are concerns that ethical debates about embryo
editing will negatively affect research on somatic cells
• we mustn’t let discussion about embryos dominate
the public debate
• We need to avoid the unhelpful ‘slippery slope’
arguments and consider the evolution of editing on a
case by case basis

Policy is on its way…
• A policy statement from the American Society of
Human Genetics on ‘Germline Gene Editing’ will be
issued shortly – has contribution from British, Canadian
and USA genetic counsellors

Audience Q&A
Please use the Question function in GoToWebinar

©PistoiaAlliance
Other CRISPR events and resources
• Pistoia Alliance member Benchling is hosting a
panel discussion: Engineering the Future:
Opportunities and Applications of CRISPR
Wednesday November 30th 5:30-7:30pm PST
Rock Health, 455 Mission Bay Boulevard, South #124, San Francisco, CA
94158 (more information at
https://www.eventbrite.com/e/engineering-the-future-opportunities-
and-applications-of-crispr-tickets-28795197210):
• Pistoia Alliance member Merck KGaA (Millipore
Sigma/SigmaAldrich) has series of technical
webinars and other videos available
http://www.sigmaaldrich.com/video/life-science/crispr-webinars.html
• Others?

©PistoiaAlliance
To address question posed by attendee re: tools
CRISPR 100November 16, 2016
• <Alvis Brazma> The two widely used tools for
CRISPR/Cas9 design that I would recommend
are:
– GPP Web Portal at the Broad Institute -
http://portals.broadinstitute.org/gpp/public/analysis-
tools/sgrna-design
– CRISPRseek Bioconductor package
http://bioconductor.org/packages/release/bioc/html/C
RISPRseek.html
• <Patrick Harrison> Recommended the following
site for additional CRISPR resources:
– https://www.addgene.org/crispr

IDMP: Overview and collaboration
opportunities
The next Pistoia Alliance Discussion Webinar:
Moderator: Gerhard Noelken
Date: January 2017
check http://www.pistoiaalliance.org/events/ for the latest information

info@pistoiaalliance.org @pistoiaalliance www.pistoiaalliance.org

CRISPR: what it is, and why it is having a profound impact on human health

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