Making genome edits in mammalian cells

HORIZON DISCOVERY

Making Genome Edits In Mammalian Cells

Chris Thorne, PhD | Commercial Marketing Manager

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Contents

1. Quick recap

2. Introducing haploid genetics

3. Observations from over 1000 knockout experiments

4. Genome editing options beyond knockouts• Knock-ins, genomic deletions, translocations, gene tagging

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The Opportunity: Genome Editing

Genome editing is the most robust and biologically relevant method for studying how genes and mutations function in driving disease

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CRISPR mediated genome editing

Exon 1 Exon 2 Exon 3

Exon Exon 2 Exon 31

CRISPR-induced DNA double-strand break

Non-homologous end joining

Exon 1

Homology-directed repair

Exon 2

Exon 2Exon 2Exon 1Frameshift mutation

Exon 1

Most frequently CRISPR-Cas9 is used to make either knockouts (via NHEJ mediated gene disruption) or knockins (via HDR)

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Cell Line

Gene Target

Guide Choice

Guide Position

Donor Design

Screening

Validation

The Key Considerations For CRISPR Gene Editing

Is it suitable?

Is it essential/expressed/amplified?

Specificity vs Efficiency

Will depend on modification

Donor design to maximise efficiency

How many clones to find a positive?

Is my engineering as expected?

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The Challenge? Polyploid cells…e.g. Disruption of the MAPK3 gene in the A375 cell line (copy number = 3)

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2

3

Validation of frameshift disruptions in polyploid cells is a significant bottleneck

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Kotecki et al. (1999) in Exp Cell ResCarette et al. (2009) in Science

KBM-7 is a human cell line that is haploid for all chromosomes but chromosome 8.

Thijn BrummelkampNKI/CeMM

The Solution? Haploid cells...

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Genotyping analysis in haploid cells


PCR with custom primers

Sanger sequencing of PCR product

Mutation masked by second copy

Mutation leads to knockout

Diploid Haploid

Both editing and validation is more efficient in haploid cells

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(Near-) Haploid Human Cell Lines

KBM-7Near-haploid (diploid chr8, chr15)Isolated from CML patientMyeloid lineageSuspension cells

HAP1Near-haploid (chr15)Derived from KBM-7Fibroblast likeAdherent cells

eHAPFully haploidDerived from HAP1Patent EP 13194940.6

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Haploid

High efficiency

Unambiguous genotyping

Diploid

Defined copy number

KnockoutsDiploid/haploid: >2fold

Defined mutationsDiploid/haploid: >10fold

Knowledge base

RNA sequencingPredict suitability as cellular model

Essentiality datasetPredict success ratefor knockouts

Advantages of haploid cells for genome editing

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Customer

Design

ProductionQualitycontrol

Packaging

Shipment

On-demand knockoutsfor any human genein 10 weeks

Production pipeline

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Knockout cell line collections

Gene CollectionsKinases, Bromodomain genes, Deubiquitinases, Ubiquitin E2 ligases, HDACs, Caspases, Rab GTPases

Pathway CollectionsSialylation, mTOR signaling, TNF- signaling, Autophagy, Epigenetics, DNA damage responses

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1500 gene targeting experiments later…

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Editing efficiency in human cells

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100

0

20

40

60

80

100

120

140

160

180

200

Editing Efficiency (in %)

# of

gRN

A pr

ojec

ts

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Cas9-induced mutational pattern

PAM-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Peak at position -3

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Cas9-induced mutational patternDeletions Insertions

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Assessment of off-target editing in clonal cell lines

Off-target sites

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Hap1 Gene Targeting – what we‘ve learned

CRISPR/Cas9 is highly efficient Mutations cluster at PAM -3

Deletions are favored over insertionsOff-target editing represents a minor issue

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So what can we do?

Exon 8 Exon 9 NanoLuc polyA

Exon 1 Exon 3

Translocations and Fusions

Gene taggingChromosomal deletions

Chr 1 Chr 19

Point mutations

Exon 8 Exon 9

*

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Exon Exon 2 Exon 31

Cas9-induceddouble-strand break

Exon 2 Exon 3

Homology-directed repair (precise)

Exon 1

Exon 1

Introduction of point mutations by homology-directed repair

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Point mutation in EGFR L858R

Targeting Efficiency ~8%

AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGAsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu

AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTGAsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeu

Clone 5

Wild-type

SpeI

Positive

contro

l

Clone 1

Clone 2

Clone 3

Clone 4

Clone 5

Clone 6

Clone 7

Clone 8

Clone 9

Clone 10

Clone 11

Clone 12

Clone 13

Clone 14

Clone 15

Clone 16

Clone 17

Clone 18

Clone 19

Clone 20

Clone 21

Clone 22

Clone 23

PCR +SpeI

Inclusion of a restriction site knockin allows rapid screening

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Chromosomal deletions

HAP1 cells are disomic for a fragment from chromosome 15

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Strategy for CRISPR/Cas-mediated excision of chr15 fragment

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Deletion of chr15 fragment is detectable by PCR

400 clones screened

5 positive clones identified

~1% targeting efficiencyEssletzbichler et al Genome Research 2014

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Single cell clones that carry the deletion can be isolated

SKY staining of clone E9

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Translocations / Chromosomal Fusions

Chin J Cancer. 2013 Nov;32(11):594-603

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Interchromosomal translocation leads to CD74-ROS1 fusion

Chr 5

Chr 6

Chr 5

Chr 6

ROS1-CD74

CD74-ROS1

Translocation

CD74

ROS1

ex6 ex7

Chr 5

Chr 6

Simultaneous cleavage with Cas9

ex33 ex34

ex7

ex6

Screen for fusion by PCR

ex33

ex34

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PCR screening identifies two clones with CD74-ROS1 fusion

CD74-ROS1

ROS1-CD74

A10

E4

E4

A10

~1% Clones Tested are positive for

fusion

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Validation of both DNA and RNA

Analysis of CD74-ROS1 break point in chr6 of genomic DNA

CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAACTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAACTTACGCATACTGCTGACAGTTAAATTTAGTTGAAGTGCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA

Predicted1C21G13

ROS1 CD74

CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAATCCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAATCCTGAAGTAGAAGGTCAAAGGGCCACCCTC------TGGATTACTTAATCCCTCTCTGAAATACCCACAAT

Predicted1C21G13

CD74 ROS1

HCT116

eHAP1C2

1G13wate

rHCT116

eHAP1C2

1G13wate

rHCT116

eHAP1C2

1G13wate

rHCT116

eHAP1C2

1G13wate

r

CD74-ROS1 ROS1-CD74 CD74 ROS1

CD74 exon 6 ROS1 exon 34

Analysis of expression of CD74-ROS1 fusion transcript

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So where next?

Exon 8 Exon 9 NanoLuc polyA

Exon 1 Exon 3

Translocations and Fusions

Gene taggingChromosomal deletions

Chr 1 Chr 19

Point mutations

Exon 8 Exon 9

*

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Gene Tagging - The conventional approach

Gene tagging by homology-directed repair


polyANanoLuc


Homology-directed repair

polyANanoLuc

Exon 9

Genome

Homology donor

Two major shortcomings: a. Low overall efficiencyb. Requires the synthesis of gene-specific donor templates

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Gene tagging by non-homologous end joining

Developed further by Thijn Brummelkamp (NKI) and Daniel Lackner (Horizon)

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Based on generic donor cassettes flanked by tia11 guide RNA recognition sites

polyANanoLuc tia11tia11

tia11 gRNAU6

Cas9

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Based on generic donor cassettes flanked by tia11 guide RNA recognition sites

Exon 7 Exon 8 Exon 9 polyANanoLuctia11


Generic donor cassettes

Non-homologous end joining (imprecise)

polyANanoLuc

tia11

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Genotyping on pools of cells after transfection

gRNA 2655

---2656

--- 2657

2658

--- 2659

2660

2661

--- 2662

--- ---2663

2665

2664

2666

2667

--- 669

---

ID1 MX2 IRF9 STAT1 TAP2 CCL2 IL9

13 out of 14 pools show integration of reporter cassette in right orientation

Exon 9 polyANanoLuc

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Single clones bearing reporter constructs

Gene ID gRNA ID # clones # PCR-positive clones

Integration confirmed by sequencing

Editing Efficiency

ID1 2655 24 3 2 8%

ID1 2656 24 5 5 21%

IRF9 2659 24 1 1 4%IRF9 2660 24 1 0 N/A

TAP2 2663 24 0 0 N/A

TAP1 2664 24 0 0 N/A

CCL2 2665 24 0 0 N/A

CCL2 2666 24 1 1 4%

IL6 996 24 3 3 13%

BUT... only one clone contained an in-frame cassette integration!

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Sequencing of individual clones

>2655-13 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2655-17 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2656-07 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC




>669-14 CTGACCCAACCACAAATGCCAGCCTGCTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2659-08 CAGATGGAGCAGGCCTTTGCCCGATACTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2666-10 CAGAAGTGGGTTCAGGATTCCATGGACCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>669-24 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC

>669-12 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC

>2656-24 CGGTCCGGGCTCCGCTCAGCACCCTCAATCCAGGGGCAGCGGATCCATGGTCTTCACACTC

Genomic Sequence Cassette Sequence

Precise cleavageLigationNo indels

Imprecise cleavageLigationIndels

Insertion is much more precise than originally predicted

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Assessing off-target integration of reporter cassette

HAP1 NanoLuc cell lines contain single integration events (as assessed by droplet digital PCR)

Hap1ID1-NanoLuc

HAP2DACT1-NanoLuc

HCT116HK2-NanoLuc

HAP1wt

NanoLuc copy number:

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A DACT1-NanoLuc reporter line

DACT1 expression is up-regulated in response to stimulation with Activin A

0

500

1000

1500

0

2000

4000

6000

8000

10000

Rela

tive

luni

nesc

ence

DACT1-NanoLuc levels

Rela

tive

luni

nesc

ence

Activin A(ng/ml)

0 10 10050 0 10 10050

4 h stimulation 24 h stimulation

Daniel Lackner

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The combination of CRISPR and a haploid background lends itself to both simple and complex genomic modifications

Modification Targeting Efficiency in Hap1Knockout >40%Point Mutation ~8%Chromosomal Deletion ~1%Chromosomal Translocation ~1%NHEJ Ligation Gene Tagging Up to 21%

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So lets wrap up!

Yes, CRISPR-Cas9 genome editing can be…

Easy to design

Efficient

Widely applicable

Flexible

…so how can Horizon help?

But…

× Not every cell line is easy to target

× Not every guide is active

× Genome editing is labour intensive and will not always be successful

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Already available…• Knockouts for >1,500 human genes• Verified by Sanger sequencing• Two independent clones per gene available• Can be supplied with gRNA used in generation• Supplied with wild type control line

$990 per cell line (Academic pricing)

How can Horizon advance your research?

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How can Horizon advance your research?

Haploid Genome Editing On Demand

• Hap1 cell line background

• Rapid, cost effective knockouts and genomic deletions

• Custom modifications also available (knockins, translocations, tags)

Custom Cell Line Engineering Service

• Your cell line

• Your choice of modification

• Fully custom service

iPSC Gene Editing Service

• Knockouts, knockins, mutation corrections

• You supply the iPSCs

• Custom modifications in 12-18 weeks

In vivo Genome Editing

• Many mice and rat knockout models already available

• Microinjection ready guide RNAs

• Custom in vivo genome editing service also available

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

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 Thorne, PhDCommercial Marketing [email protected] +44 1223 204 799

Follow me on LinkedIn: cmcthorne

mailto:[email protected]