lessons learned from high throughput crispr targeting in human cell lines

HORIZON DISCOVERY

Genome Editing Comes of Age

Lessons learned from high throughput CRISPR targeting in human cell lines

Chris Thorne, PhD | Commercial Marketing Manager

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Overview

Introduction to genome editing and CRISPR-Cas9

Haploid cells – the genome editors dream (and lessons learned from 1500 experiments)

High throughput genome editing – where next?

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The Genomic Era…

Adapted from The US National Human Genome Research Institute, (2003) Nature

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The Genomic Era…

1. Elucidate the organisation of

genetic networks and their

contribution to cellular and

organismal phenotypes

2. Understand the heritable

variations and their association

with health and disease

3. Translate genome-based

knowledge into health benefits


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Gene function analysis - Patient-derived cell lines

Human cell lines contain pre-existing mutations

are derived directly from human tumors

Immense genetic diversity

HoweverLack 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)

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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

Total overlap

only 3 genes

Shalem et al Science 2014 HIV Host Factors

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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

However

Difficult to achieve long-term overexpression

• Large growth induction phenotype• Transforming alone

• Milder growth induction phenotype• Non-transforming alone

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The Opportunity: Genome Editing

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The Opportunity: Genome Editing

1. Elucidate the organisation of

genetic networks and their

contribution to cellular and

organismal phenotypes

Knockouts

2. Understand the heritable

variations and their association

with health and disease

Knock-ins

3. Translate genome-based

knowledge into health benefits

Gene Therapy


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CRISPR/Cas system: Adaptive immunity in bacteria

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The CRISPR/Cas revolution

Jinek et al. (2012) in ScienceCong et al. (2013) in ScienceMali et al. (2013) in ScienceCho et al. (2013) in Nat Biotech

AGCTGGGATCAACTATAGCG CGG

gRNA target sequence PAM

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CRISPR mediated genome editing

Exon 1 Exon 2 Exon 3

Exon Exon 2 Exon 31

Cas9 nuclease-induced DNA double-strand break

Non-homologous end joining

Exon 1

Homology-directed repair

Exon 2

Exon 2Exon 2Exon 1

Frameshift mutation

Exon 1

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Cell Line

Gene Target

Guide Choice

Guide Position

Donor Design

Screening

Validation

Challenges – Experimental Design

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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Challenges - Polyploid cells…

e.g. Disruption of the MAPK3 gene in the A375 cell line (copy number = 3)

1

2

3

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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

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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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Advantages of haploid cells for genome editing

High efficiency

Unambiguous genotyping

Defined copy number

Knockouts>2 fold improvement

Defined mutations>10 fold improvement

Knowledge base

RNA sequencingPredict suitability as cellular model

Essentiality datasetPredict success ratefor knockouts

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Production pipeline

Shipment

Packaging

Quality Control Production

Design

Customer

Custom knockouts for any human gene

in 10 weeks

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Available gene sets

Gene sets in the making• Phosphoinositide Metabolism (~50)• Phospholipases (~25)• Protein Phosphatases (~90)• TRIM Ubiquitin E3 ligases (~70)• FDA drug targets (~300)

Available collection• Knockouts for >1,500 human genes• Verified by Sanger sequencing• One gRNA per gene• Two clones per gRNA

www.horizondiscovery.com/cell-lines

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1500 gene targeting experiments later…

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Editing efficiency in human cells

0

20

40

60

80

100

120

140

160

180

200#

of

gRN

A p

roje

cts

Editing Efficiency (in %)

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Cas9-induced mutational pattern

PAM

0

200

400

600

800

1000

1200

1400

1600

1800

2000

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

Peak at position -3

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Cas9-induced mutational pattern

Deletions 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 insertions

Off-target editing represents a minor issue

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Cell line engineering in haploid cells

Knockouts Deletions Translocations

Point mutations Insertions Reporters

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Haploid Knockin Cell Lines

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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%

AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTG

AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu

AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTG

AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeuClone 5

Wild-type

SpeI

PCR +SpeI

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Chromosomal Deletions

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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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Genomic MALAT1 deletion leads to loss of MALAT1 RNA

RNA/ cDNAGenomic DNA

Deletion PCR MALAT1 PCR MALAT1 RT-PCR GAPDH RT-PCR

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Chromosomal Translocations

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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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Clones 1C2 and 1G13 contain the CD74-ROS1

CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA

CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA

CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAGTGCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA

CD74-ROS1 (chr 6)

PredictedClone 1C2Clone 1G13

ROS1 CD74

CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAAT

CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAAT

CCTGAAGTAGAAGGTCAAAGGGCCACCCTC------TGGATTACTTAATCCCTCTCTGAAATACCCACAAT

PredictedClone 1C2Clone 1G13

CD74 ROS1

CD74-ROS1 ROS1-CD74 CD74 ROS1

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CD74-ROS1 fusion is expressed in clone 1G13

RT-PCR from clone 1G13 shows the CD74-ROS1 fusion transcript

CD74 exon 6 ROS1 exon 34

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Engineering of EML4-ALK fusionEML4 ALK

EML4-ALK

Inversion

Genomic DNA

Chr 2

Chr 2

RNA/cDNA

gRNA2gRNA1

EML4 ALK

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Reporter cell lines

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The conventional approach

Gene tagging by homology-directed repair


Reporter


Homology-directedrepair

Reporter

Exon 9

Genome

Homology donor

Major shortcoming: 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 Horizon Discovery

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Gene tagging by non-homologous end joining

Cas9 cleavage and ligation by NHEJ

Exon 8 Exon 9

gRNA

Gene-specific gRNA

Tagtia11 tia11

tia11 gRNA

tia11 gRNAU6

Exon 8 Exon 9 Tag

Generic tagging plasmid

Tagged gene at endogenous locus

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Genotyping on pools of cells after transfection

Exon NanoLuc®

gRNA

ID1 MX2 IRF9 STAT1 TAP2 CCL2 IL6

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

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Generation of single clones

Successful recovery of single clones for 5 out of 9 cell lines

Gene gRNA IDTagged Clones/Total

Clones Editing Efficiency (%)

ID1 2655 2/24 8.3

ID1 2656 5/24 20.8

IRF9 2659 1/24 4%

IRF9 2660 0/24 N/A

TAP2 2663 0/24 N/A

TAP1 2664 0/24 N/A

CCL2 2665 0/24 N/A

CCL2 2666 1/24 4%

IL6 669 3/24 13%

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

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Sequencing of individual clones

Genomic locus

GAAGTTGGAACCCCCGGGGGCCGAGGGCTGCCGGTCCGGGCTCCGCTCAGCACCCTCAACGGCGAGATCAGCGCCCTGAC

Reporter cassette

GGTACTTCGCGAATGCGTCGAGATGAATTCGGTATGTCGGGAACCTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

Resulting clone 2655-13

ACTCGGAATCCGAAGTTGGAACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

gRNA 2655

gRNA tia11

Exon 7 Exon 8 Exon 9 NanoLuc® tiatia

Self-liberating reporter cassette

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Most clones show precise ligation at PAM minus 3 without Indels

>2655-13 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2655-17 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2656-07 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC




>669-14 CTGACCCAACCACAAATGCCAGCCTGCTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2659-08 CAGATGGAGCAGGCCTTTGCCCGATACTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>2666-10 CAGAAGTGGGTTCAGGATTCCATGGACCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC

>669-24 CTGACCCAACCACAAATGCCAGCCTGCT-------GCAGCGGATCCATGGTCTTCACACTC

>669-12 CTGACCCAACCACAAATGCCAGCCTGCT-------GCAGCGGATCCATGGTCTTCACACTC

>2656-24 CGGTCCGGGCTCCGCTCAGCACCCTCAATCCAGGGGCAGCGGATCCATGGTCTTCACACTC

Genomic Sequence Cassette Sequence

Imprecise cleavage/ligationIndels

Precise cleavage/ligationNo indels

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Second generation tagging cassette

NanoLuc®tia11 tia11

No start or stop codon: insert anywhere in the gene

Three versions: one for each reading frame

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NanoLuc® reporter cell lines

• Tag 3 cytokine-responsive genes at the 3‘ end with NanoLuc cassette• Genotyping of single clones

Exon NanoLuc® Exon

PCR 5’ junction PCR 3’ junction

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NanoLuc® reporter cell lines are functional

• Stimulate tagged cell lines with cytokines • Measure change in protein levels by luciferase assay

NanoLuc® NanoLuc®

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TurboGFP reporter cell lines

• Tag endogenous genes with TurboGFP cassette• Enrichment for targeted clones by FACS• Genotyping single clones

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Localization of TurboGFP-tagged proteins

Assess subcellular localization by microscopy

TERF1LMNA

TurboGFP

DAPI

Enlarged(merged)

TERF1

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13 out of 14 clones contain single integration events

Assessing off-target integration of reporter cassette

Copy number determination using Droplet Digital PCR

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Summary

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

Modification Targeting Efficiency in Hap1

Knockout >40%

Point Mutation ~8%

Chromosomal Deletion ~1%

Chromosomal Translocation ~1%

NHEJ Ligation Gene Tagging Up to 21%

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Acknowledgements

Academic CollaboratorsThijn Brummelkamp (NKI)Bill Skarnes (Sanger)Jin-Soo Kim (Seoul)

Horizon Vienna TeamDaniel LacknerTilmann BürckstümmerPaloma Guzzardo

Horizon Cambridge TeamPhilippe CollinDavid HughesSergey Lekomtsev

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Available gene sets

Gene sets in the making• Phosphoinositide Metabolism (~50)• Phospholipases (~25)• Protein Phosphatases (~90)• TRIM Ubiquitin E3 ligases (~70)• FDA drug targets (~300)

Available collection• Knockouts for >1,500 human genes• Verified by Sanger sequencing• One gRNA per gene• Two clones per gRNA

www.horizondiscovery.com/cell-lines

On demand modifications• Knockouts• Deletions• Knockins• Translocations• Endogenous tags

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, PhD

Commercial Marketing Manager

[email protected]

+44 1223 204 799

mailto:[email protected]