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

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CRISPR 4

The Panel

Patrick Harrison, Senior Lecturer Physiology, University College of CorkThe 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-EBIDr 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, AmgenDr. 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

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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CRISPR 5

Agenda

• 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 – Cas9 Gene EditingWhy How What

Patrick Harrison, PhD – University College Cork, Ireland

7CRISPR

Val His Leu Thr Pro Glu Glu Lys Ser Asp

November 16, 2016

8CRISPR

Val His Leu Thr Pro Val Glu Lys Ser AspTarget DNA

November 16, 2016

9CRISPR

Thr Pro Glu Glu Lys

Val His Leu Thr Pro Val Glu Lys Ser AspTarget DNA

Donor DNA

November 16, 2016

10CRISPR

Target DNA

Donor DNA

Clustered Regularly Interspaced Short Palindromic Repeats

Cas9

guide RNA

November 16, 2016

11CRISPR

Target DNA

Donor DNA

Cas9

guide RNA

Cut Resect Incorporate Seal Precision Repair

November 16, 2016

12CRISPR

Target DNA

Donor DNA

Cas9

guide RNA


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13CRISPR


November 16, 2016

14CRISPR


November 16, 2016

15CRISPR


November 16, 2016

16CRISPR


November 16, 2016

17CRISPR


November 16, 2016

18CRISPR

Thr Pro Glu Glu Lys

Val His Leu Thr Pro Glu Glu Lys Ser Asp


HDRHomology-directed repair

November 16, 2016

19CRISPR

Target DNA

Cas9

guide RNA

November 16, 2016

20CRISPRNovember 16, 2016

21CRISPRNovember 16, 2016

CRISPR

Val His Leu Thr Pro Gly Glu Val STOP

NHEJNon-homologous

end joining

In-frame STOP codon = gene knock-out

November 16, 2016 22

CRISPR

Cas9(2013)

gRNA

Why has Cas9/gRNA surpassed ZFNs and TALENs?

ZFNs(1996)

TALENs(2011)

Standing on the shoulders of giants


24CRISPR

Cas9/guideRNADesign and synthesis (2013)

November 16, 2016

25CRISPR

|||||||||||||||||||| 5’ GUCACCUCCAAUCAGUAGGG 3'

gRNA


November 16, 2016

26CRISPR

|||||||||||||||||||| 5’ GUCACCUCCAAUCAGUAGGGGUUUUAGAGCUAG .|||||. |||| GUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAUA |||| GAA AAAGUGGCACCGA .|||||||G 3’ UUUUUUCGUGGCU

AA

AA

gRNA


November 16, 2016

27CRISPR

|||||||||||||||||||| 5’ GUCACCUCCAAUCAGUAGGGGUUUUAGAGCUAG .|||||. |||| GUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAUA |||| GAA AAAGUGGCACCGA .|||||||G 3’ UUUUUUCGUGGCU

AA

AA

Cas9

DSB

gRNA


November 16, 2016

28CRISPR

Double Stranded BreakTwo 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

29CRISPR

GFP transgenic

rat

~40% Knock Outs

CRISPR 2016

MiceRatsCowsSheepRabbits

MonkeysZebrafish

Human Embryos (14-day limit)

Transfer KO eggsto surrogatePronuclei

Germline EditingInject Nuclease into Fertilised Eggs

Yang 2009

November 16, 2016

30CRISPR

F.IX genevariant 1 2 3 4

Pre-clinical in vivo editing – haemophilia Bi.v. inject nuclease and donor

Li 2011

November 16, 2016

31CRISPR



Li 2011

November 16, 2016

32CRISPR



Li 2011

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33CRISPR

F.IX genevariant

Super-exon Donor

1 2 3 4

2 3 4

F.IX genewild-type

1 2 3 42 3 4

Li 2011

November 16, 2016

34CRISPR

Gene Editing = Permanent

cDNA addition = Transient

Li 2011

November 16, 2016

35CRISPR



Li 2011

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36CRISPR



Li 2011

Li 2011

November 16, 2016

37CRISPR

100 to 1,000-fold reduction in viral load

Control

Tebas 2014

Patient #1ZFNs delete CCR5

November 16, 2016

38CRISPR

Acute Lymphoid Leukaemia

Modified Patient T cells

CAR targets CD19

TALENs delete TCR (avoids rejection)

2015 Patient #2TALENs enable CAR-T cells

November 16, 2016

39CRISPR

CRISPR – Cas9 Gene EditingInterim 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

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

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CRISPR 42

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

November 16, 2016

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CRISPR 43

Gene Knockout & Editing by CRISPR/Cas9

Figure from ThermoFisher

November 16, 2016

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CRISPR

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 sgRNACells+Cas9/target sgRNA

Target protein expression levelCe

ll co

unt


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CRISPR 45

Rapid Gene Knockout by Delivery of Cas9/sgRNA Ribonucleoprotein complex

Not-so-rapid clonal isolation of edited cells

Ran et al, Nature Protocols, 2013Liang et al, J Biotech, 2015

November 16, 2016

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CRISPR

Pooled Lentiviral sgRNA Libraries for Genome-scale Gene Knockout Screening

Figure adapted from Hartenian & Doench, FEBS, 2015


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Beyond Gene Knockout: Alternative Cas9-mediated Screening Methods

Sanjana, Analytical Biochem., 2016

47November 16, 2016 CRISPR

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 challengeFrom predicting the target sites to assessing the effects

Alvis Brazma – EMBL-EBI

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CRISPR 50

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

November 16, 2016

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CRISPR 51

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

November 16, 2016

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CRISPR 52

Selecting the sgRNA sequence

Genome

Gene of interest

20 nt sequence

Most similar sequence off target

Target sequence - perfect match


TCC

November 16, 2016

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CRISPR 53

Selecting the sgRNA sequence

Genome

Gene of interest

20 nt sequence



Target sequence - perfect match

TCC TCC

November 16, 2016

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54CRISPR

Annotated CRISPR/Cas9 target sites in Ensembl genome browser at European Bioinformatics Institute

Anna Farne, Mark Thomas, David Parry-Smith

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CRISPR 55

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

November 16, 2016

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56CRISPR

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

November 16, 2016

Cystic FibrosisThe Case for Gene Editing

Patrick Harrison, Ph.D. – University College Cork, Ireland


Cl-Cl-

HCO3-

HCO3-

Airway hydrationNeutral pH

Normal LungCFTR anion channel

Cilia beating

H2O H2O


Cilia beating

CF LungMutations in CFTR gene

collapsed

Cl-Cl-

HCO3-

HCO3-

H2O H2O

Cilia collapsed


Class IIINo conductance

Class IIReduced Trafficking

Class INo protein

CF – Personalised Medicine

4,000 people 92,000 people 4,000 people


Abs

olut

e ch

ange

in %

of

pre

dict

ed F

EV1

Ramsay 2011

Nick Talbot

Class IIINo conductance

4,000 people


Abs

olut

e ch

ange

in %

of

pre

dict

ed F

EV1

.

Wainwright 2015

Orkambi (dose A)

Orkambi (dose B)

Placebo

BA

Class IIReduced Trafficking

92,000 people


Abs

olut

e ch

ange

in %

of

pre

dict

ed F

EV1

cDNA

Placebo

Alton 2015

Multi-dose CFTR cDNA is safe

Class INo protein

4,000 people

64CRISPR

Genome F508del5'3'

3’5'

Donor CTT 3'5'

5'3'

CTT GAA

------

Lee 2012

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65CRISPR

Genome F508del5'3'

3’5'

Donor CTT 3'5'

5'3'

CTT GAA

------

Lee 2012

November 16, 2016

66CRISPR

Genome F508del5'3'

3’5'

NGG

Donor CTT 3'5'

5'3'

CTT GAA

------

Schwank 2013

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67CRISPR

5'3'

3’5'

3'5'

5'3'

CTT GAA

Genome F508del

Donor CTT

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68CRISPR

5'3'

3’5'

3'5'

5'3'

GAA

CTT

Genome F508del

Donor CTT

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69CRISPR

5'3'

3’5'

3'5'

5'3'

GAA

CTT

Genome F508del

Donor CTT

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70CRISPR

5'3'

3’5'

3'5'

5'3'

GAA

CTT

CTT

Genome F508del

Donor CTT

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71CRISPR

5'3'

3’5'

3'5'

5'3'

GAA

CTT

CTTGAA

CTT

Genome F508del

Donor CTT

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72

Editing restores function Stem Cell Organoids

WT F508del F508del edited

November 16, 2016 CRISPR

Schwank 201372

73




Schwank 201373

74




Schwank 201374

75

Schwank 2013




75

76CRISPR

HDRrestores

CFTR<1%

Select & enrich

NHEJ >50%KO

How can KO fix CF?

76

November 16, 2016

77CRISPR

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) mutationsDeep intron Mutations

3272 -26A>G(n = 463)

3849 +10kb C>T(n = 1,143)

* * *1811+1.6kb A>G

(n = 71)

77

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78CRISPR

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

November 16, 2016

79CRISPR

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

November 16, 2016

80CRISPR

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

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81CRISPR

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

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82CRISPR

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

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One for AllCFTR super-exons

83

84CRISPR

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

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85CRISPR

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

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86CRISPR

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 201686

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87CRISPR

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 201687

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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 11-27CFTR Exons 11-27

3’

Bednarski 201688


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

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Cystic Fibrosis – Cas9 Gene EditingInterim Summary

• HDR – precise but inefficient

• NHEJ – efficient but only 2% of individuals

• HDR superexon – all mutations but inefficient

• NHEJ superexon – TBC

Cystic FibrosisThe Case for Gene Editing

Patrick Harrison, Ph.D. – University College Cork, Ireland

Ethics and CRISPRDr Anna Middleton

Head of Society and Ethics ResearchWellcome 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&APlease use the Question function in GoToWebinar

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Other CRISPR events and resources

• Pistoia Alliance member Benchling is hosting a panel discussion: Engineering the Future: Opportunities and Applications of CRISPRWednesday November 30th 5:30-7:30pm PSTRock 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 availablehttp://www.sigmaaldrich.com/video/life-science/crispr-webinars.html

• Others?

Partner logo if required

https://www.eventbrite.com/e/engineering-the-future-opportunities-and-applications-of-crispr-tickets-28795197210

https://www.eventbrite.com/e/engineering-the-future-opportunities-and-applications-of-crispr-tickets-28795197210

http://www.sigmaaldrich.com/video/life-science/crispr-webinars.html

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CRISPR 100

To address question posed by attendee re: tools

November 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 packagehttp://bioconductor.org/packages/release/bioc/html/CRISPRseek.html

• <Patrick Harrison> Recommended the following site for additional CRISPR resources:– https://www.addgene.org/crispr



http://bioconductor.org/packages/release/bioc/html/CRISPRseek.html

http://bioconductor.org/packages/release/bioc/html/CRISPRseek.html

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

http://www.pistoiaalliance.org/events/

[email protected] @pistoiaalliance www.pistoiaalliance.org

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

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