CRISPR: what it is, and why it is having a profound impact on human health
1. 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
3. 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
18. Thr Pro Glu Glu Lys
Val His Leu Thr Pro Glu Glu Lys Ser Asp
Cut Resect Incorporate Seal Precision Repair
HDR
Homology-directed repair
November 16, 2016 CRISPR 18
22. 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
23. 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
28. 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
November 16, 2016 CRISPR 28
29. 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
November 16, 2016 CRISPR 29
30. F.IX gene
variant
1 2 3 4
Pre-clinical in vivo editing – haemophilia B
i.v. inject nuclease and donor
Li 2011
November 16, 2016 CRISPR 30
31. F.IX gene
variant
1 2 3 4
Pre-clinical in vivo editing – haemophilia B
i.v. inject nuclease and donor
Li 2011
November 16, 2016 CRISPR 31
32. F.IX gene
variant
1 2 3 4
Pre-clinical in vivo editing – haemophilia B
i.v. inject nuclease and donor
Li 2011
November 16, 2016 CRISPR 32
37. 100 to 1,000-fold
reduction in viral
load
Control
Tebas 2014
Patient #1
ZFNs delete CCR5
November 16, 2016 CRISPR 37
38. Acute Lymphoid Leukaemia
Modified Patient T cells
CAR targets CD19
TALENs delete TCR
(avoids rejection)
2015 Patient #2
TALENs enable CAR-T cells
November 16, 2016 CRISPR 38
39. 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?
November 16, 2016 CRISPR 39
40. 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
48. 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
60. 60November 16, 2016 CRISPR
Class III
No conductance
Class II
Reduced Trafficking
Class I
No protein
CF – Personalised Medicine
4,000 people 92,000 people 4,000 people
61. 61November 16, 2016 CRISPR
Absolutechangein%
ofpredictedFEV1
Ramsay 2011
Nick Talbot
Class III
No conductance
4,000 people
62. 62November 16, 2016 CRISPR
Absolutechangein%
ofpredictedFEV1
.
Wainwright 2015
Orkambi (dose A)
Orkambi (dose B)
Placebo
BA
Class II
Reduced Trafficking
92,000 people
63. 63November 16, 2016 CRISPR
Absolutechangein%
ofpredictedFEV1
cDNA
Placebo
Alton 2015
Multi-dose
CFTR cDNA is
safe
Class I
No protein
4,000 people
90. November 16, 2016 CRISPR 90
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
91. Cystic Fibrosis
The Case for Gene Editing
Patrick Harrison, Ph.D. – University College Cork, Ireland
92. Dr Anna Middleton
Head of Society and Ethics Research
Wellcome Genome Campus
Cambridge, UK
November 16, 2016 CRISPR 92
93. 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
November 16, 2016 CRISPR 93
94. 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?
November 16, 2016 CRISPR 94
95. 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
November 16, 2016 CRISPR 95
96. 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
November 16, 2016 CRISPR 96
97. 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
November 16, 2016 CRISPR 97
101. 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