genome editing comes of age

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HORIZON DISCOVERY Genome Editing Comes of Age CRISPR, rAAV and the new landscape of molecular cell biology Chris Thorne | Gene Editing Specialist

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HORIZON DISCOVERY

Genome Editing Comes of AgeCRISPR, rAAV and the new landscape of molecular cell biology

Chris Thorne | Gene Editing Specialist

2

Horizon Discovery

Powering Genomic Research and Translational Medicine,

…from Sequence to Treatment

3

Products/Services

4

INDUSTRY ACADEMIA

Our Customers: Over 1,000 customers in more than 50 countries

5

Overview

• The case for Genome Editing

• Genome Editing Tools• rAAV• CRISPR/Cas9

• Key Considerations for Gene Editing

• Genome Editing at scale• High through Knock-out Cell Line Generation• Genome Wide sgRNA Synthetic Lethality Screening

5

6

The Opportunity:

The challenge has shifted from obtaining genomic information to understanding what it means

7

Gene function analysis - Patient-derived cell lines

Human cell lines contain pre-existing mutations

are derived directly from human tumors

Immense genetic diversity

However Lack of wild type controls

Availability of rare mutation models

Cell line diversity makes it very hard link observations to specific genetics

(Domke et al Nat. Comms 2013)

8

Gene function analysis - RNAi

Problems with RNAi can result in false positives or negatives

Loss of function analysis using RNAi is

inexpensive and widely applicable

Incomplete knockdown

However Lack of reproducibility

Off-target effects

Brass et al.Science

273 genes

König et al.

Cell

213 genes

Zhou

et a

l.

Cell H

ost M

icrob

e

300

gene

s

Total overlap only 3 genes

Shalem et al Science 2014 HIV Host Factors

9

Gene function analysis - Overexpression

Overexpression of oncogenes can over represent their role in disease biology

Gain of function analysis using

overexpression is inexpensive and widely

applicable

Result may be artefact of overexpression

HoweverDifficult to achieve long-

term overexpression

• Large growth induction phenotype• Transforming alone

• Milder growth induction phenotype• Non-transforming alone

10

Gene function analysis – Gene Editing

Gene editing represents the most biologically relevant means to explore gene function in cells

Genetically defined mutant cell line

Matched isogenic wild type control

Complete loss of function possible

Endogenous gain of function possible

11

Genome Editing: The Right Tool For The Right Outcome

Horizon is ‘technology agnostic’, using the right technology to

generate virtually any genomic modification

in a cell

12

The Right Tool For The Right Outcome

rAAV• High precision / low thru-put• Any locus, wide cell tropism• Well validated, KI focus• Exclusive to HD

Zinc Fingers • Med precision / med thru-put• Good genome coverage• Well validated / KO Focus• Licensed from Sigma

CRISPR • New but high potential• Capable of multi-gene targeting• Simple RNA-directed cleavage• Combinable with AAV• Extensive IP position

Great for knock-outsGreat for heterozygous knock-ins

13

rAAV: How Does It Work?

Nature Genetics 18, 325 - 330 (1998)

AAV = Adeno Associated Virus (ssDNA)

14

Crispr (cr) RNA + trans-activating (tra) crRNA combined = single guide (sg) RNA

CRISPR/Cas9: How Does It Work?

15

AGCTGGGATCAACTATAGCG CGGgRNA target sequence PAM

CRISPR/Cas9: How Does It Work?

16

Cas9 Wild type Cas9 Nickase (Cas9n)

Induces double strand break Only “nicks” a single strand

Only requires single gRNA Requires two guide RNAs for reasonable activity

Concerns about off-target specificity Reduced likelihood of off-target events

High efficiency of cleavage Especially good for random indels (= KO)

Guide efficiency dictated by efficiency of the weakest gRNA

CRISPR/Cas9: How Does It Work?

Nishimasu et al Cell

17

CRISPR/Cas9: What can you do?

Hsu et al. Cell. 2014

18

Genome Editing: As Simple As…

... HOWEVER …

Cell Line

Screen for clones

Engineered cells!

Genome Editing Vector

19

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

20

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Normal human karyotype

HeLa cell karyotype

Gene copy number Effect of modification on growth

21

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Transfection/electroporation Single-cell dilution

22

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Sequence source Off-target potential Guide proximity Wild-type Cas9 or mutant nickase

23

Ran et al Cell 2014

24

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Number of gRNAs gRNA activity measurement

NTCas9 wt

only4uncut 1 52 3

gRNA

200

300

400

500

100

600

+ve

700

200

300

400

500

100

600700

25

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Donor sequence modifications Modification effects on expression or splicing Size and type of donor (AAV, oligo, plasmid) Selection based strategies

Cas9 Cut Site

Genomic Sequence

Donor Sequence containing mutation

26

Technology Development at Horizon: Systematic improvementsDonor lengths: sODNs ranging from 50-200nt, with single phosthothioate modifications at both outer nucleotides

100nt ssODN is optimal

40 60 80 100 120 140 160 180 2000.0

0.5

1.0

1.5

HR efficiency using ssODNs of different lengths

Oligo length (NT)

Effi

cien

cy (

%)

40 60 80 100 120 140 160 180 2000

5

10

15

Transfection efficiency using 10 pmol ssODN

Oligo length (NT)

Tra

nsfe

ctio

n %

(R

FP

)

Size Oligo Sequence50 C*ACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCC*C70 T*CCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTG*C90 T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A

110 A*CAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG*C130 T*TTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC*G150 G*TATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA*A170 T*AAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAA*C200 A*AATGTCTTTATAAATAAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA*C

GFP Mutation, PAM mutation

27

Technology Development at Horizon: Systematic improvementsDonor modifications: number and position of phosphothioate medications

Only 3’ PTO modifications required for ssODNs tested

Oligo Sequence

None TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA5' PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A

5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*AMut Flank TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA

Mut Flank + 5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A3x5' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA3x3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A

3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAMut Flank + 3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A

GFP Mutation, PAM mutation

None

5' P

TO

3' P

TO

5+3

PTO

Mut F

lank

Mut F

lank

+ 5+

3 PTO

3x5'

PTO

3x3'

PTO

3x5'

+3' P

TO

Mut F

lank

+ 3x

5'+3

' PTO

0.0

0.5

1.0

Ta

rge

tin

g f

req

ue

nc

y (

GF

P%

)

HR efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides

None

5' P

TO

3' P

TO

5+3

PTO

Mut F

lank

Mut F

lank

+ 5+

3 PTO

3x5'

PTO

3x3'

PTO

3x5+

3 PTO

Mut F

lank

+ 3x5

+3 P

TO

0

5

10

15

20

25

Tra

nsfe

ctio

n %

(R

FP

)

Transfection efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides

None

5' P

TO

3' P

TO

5+3

PTO

Mut F

lank

Mut F

lank

+ 5+

3 PTO

3x5'

PTO

3x3'

PTO

3x5'

+3' P

TO

Mut F

lank

+ 3x

5'+3

' PTO

0.0

0.5

1.0

Ta

rge

tin

g f

req

ue

nc

y (

GF

P%

)

HR efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides

None

5' P

TO

3' P

TO

5+3

PTO

Mut F

lank

Mut F

lank

+ 5+

3 PTO

3x5'

PTO

3x3'

PTO

3x5+

3 PTO

Mut F

lank

+ 3x5

+3 P

TO

0

5

10

15

20

25

Tra

nsfe

ctio

n %

(R

FP

)

Transfection efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides

28

Introducing gUIDEBook™

Supports all Cas9 nuclease variants

Advanced tools for knock-in design

Comprehensive gRNA scoring • Off target• Activity

Full integration with annotated reference genomes

Flexible and easy to use

29

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Donor sequence modifications Modification effects on expression or splicing Size and type of donor (AAV, oligo, plasmid) Selection based strategies

(+/+)

(+/-)

(-/-)

(KI/-)

(KI/+)(KI/KI)

30

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Number of cells to screen Screening strategy Modifications on different alleles Homozygous or heterozygous

modifications versus mixed cultures

% cells targeted

31

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Confirmatory genotyping strategies Off-target site analysis Modification expression Contamination

Heterozygous knock-in

Wild type

32

Key Considerations For CRISPR Gene Editing

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

How many copies?

Is it suitable?

What’s my goal? (Precision vs Efficiency)

Does my guide cut?

Have I minimised re-cutting?

How many clones to find a positive?

Is my engineering as expected?

33

High throughput knock-out cell line generation

(Near) Haploid human cell lines

• Near-haploid (diploid for chr8, and chr15)• Isolated from CML patient• Myeloid lineage• Suspension cells

KBM-7

HAP1

• Near-haploid (diploid for chr15)• Derived from KBM-7• Fibroblast like• Adherent cells

34

Unambiguous genotyping

Defined copy number Knowledge base

RNA sequencing- Predict suitability as cellular model

Essentiality dataset- Predict success rate for knockouts

Haploid

High efficiency

Diploid

- Knockouts

- Defined mutations

High throughput knock-out cell line generation

Advantages of Haploid Cells

35

Wildtype TCCTTTGCGGAGAGCTGCAAGCCGGTGCAGCAG ||||||||||| ||||||||||||||Knockout TCCTTTGCGGA--------AGCCGGTGCAGCAG

Wildtype SerPheAlaGluSerCysLysProValGlnGlnKnockout SerPheAlaGlu AlaGlyAlaAla

Exon 1

DNA sequencing

Exon 2

Cas9 cleavage

High throughput knock-out cell line generation

CRISPR/Cas9 allows rapid and high efficiency targeting

36

Customer

Design

ProductionQualitycontrol

Packaging

Shipment

Custom knockoutsfor any human gene

in 10 weeks

High throughput knock-out cell line generation

37

Exon 7 Exon 8 Exon 9

Exon 8

Cas9-induceddouble-strand break

Non-homologous end joining (imprecise)

Exon 9Exon 7

Exon 9Exon 8Exon 7

Insertion of DNA cassettes by NHEJ

38

EEA1-GFP DAPI Merge

LAMP1-GFP DAPI Merge

KDM1A-GFP DAPI Merge

Nucleus

Lysosomes

Early Endosomes

39

Genome Wide sgRNA Screening

Lentivirally delivered sgRNA can drive efficient cleavage of target genomic

sequences for use in whole genome screens

Use massively-parallel next-gen sequencing to assess results

Possible addition/replacement to RNAi screens

40

Genome Wide sgRNA Screening

40

Shalem et al Science 2014

41

We are combining CRISPR and isogenic cell lines to perform

CRISPR-based Synthetic Lethality Screens

sgRNA technology will be transformational for both Target ID and early-stage Validation

Synthetic lethal target ID via sgRNA screening

41

42

Ready-made knock-out X-MAN® cell linesX-MAN® - gene X Mutant And Normal cell line

Advantages:• Genetically verified• More than 3,000 available clones already available, in a variety of cell line backgrounds• Quick and easy way to get first data on gene of interest• Available with validated gRNAs to use with your own human cell line of choice.

More Information: www.horizon-genomics.com/

Bromodomain40 genes

Autophagy15 genes

mTOR pathway50 genes

Kinases350 genes

HATs/HDACs15 genes

DNA damage50 genes

RAB GTPases15 genes

Deubiquitinases80 genes

43

Genome Editing Tools and Services

• Wild type and nickase• Separate or all-in one vectors

• gRNA design and validation service• Pre-validated guides available

• Custom donor design and synthesis• Multiple formats inc. rAAV available

• >1000 ready-modified cell lines• Custom cell line generation service

• Viral encapsulation of rAAV donor• Project design support

Your Horizon Contact:

t + 44 (0)1223 655580f + 44 (0)1223 655581e [email protected] www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Chris ThorneGene Editing [email protected] +44 1223 204 742

Useful Resources

From Horizon

Technical manuals for working with CRISPR - http://www.horizondiscovery.com/talk-to-us/technical-manuals

In the Literature

Exploring the importance of offset and overhand for nickase - http://www.cell.com/cell/abstract/S0092-8674(13)01015-5

sgRNA whole genome screening:• Shalem et al - http://www.sciencemag.org/content/343/6166/84.short• Wang et al - http://www.sciencemag.org/content/343/6166/80.abstract

On the web

Feng Zhang on Game Changing Therapeutic Technology (Link to Feng’s Video)

Guide design - http://crispr.mit.edu/

CRISPR Google Group - https://groups.google.com/forum/#!forum/crispr