HORIZON DISCOVERY

Resolving Ambiguity in Target ID ScreensCRISPR-Cas9 Based Essentiality Profiling

Julie Wickenden, PhD

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Pathfinder Target Essentiality Assay Service

• A new CRISPR─Cas9 based medium throughput assay service for validation of target gene essentiality

• Can be used to resolve ambiguous screening results

• Can also provide information on drug target suitability

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Target ID and Validation | RNAi

Loss of function analysis using RNAi is inexpensive and widely applicable

However

Shalem et al Science 2014

Problems with RNAi can result in false positives or negatives

shRNATotal overlap only 3 genes

HIV Host Factors

Brass et al.Science

273 genes

König et al. Cell

213 genesZhou et al.

Cell Host Microbe

300 genes

Incomplete knockdown

Lack of reproducibility

Off-target effects

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The problems with RNAi

Off-target effects of RNAi do not correlate with BLAST scores!

Off-target effects can occur in a sequence-independent manner

Sequence-related off-target effects of RNAi are driven by seed sequence

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Is there a better way to ID targets?High throughput KO screening with CRISPR-Cas9

Jinek et al. (2012) in ScienceCong et al. (2013) in Science Mali et al. (2013) in ScienceCho et al. (2013) in Nature Biotech Exploits NHEJ for KO generation

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Target Validation at Horizon | is CRISPR-Cas9 the answer?

CRISPR-Cas9 KO technology is thought to have less severe off-target effects than shRNAKnock-out approaches can reveal phenotypes that are missed by knockdown approachesCas9/sgRNA approaches to the validation of anti-proliferative targets have their own issues

Only 2/3rd of repairs will cause a frameshift mutation that leads to early termination

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Pathfinder Target Essentiality Assay

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Target Essentiality Assay | Requirements

1. The cell line selected must robustly grow from single cells

• 53 cell lines have been pre-evaluated for this assay (listed on a later slide)

• Evaluation of your cell line of choice is possible should it not feature on this list

2. The guide RNA must efficiently direct Cas-9 nuclease activity to the DNA

• If an efficient guide RNA has not been identified we can design and evaluate guides for you

3. The standard assay format is for cell lines where the copy number of the target gene is 2-3

• Should the copy number of your gene of interest be higher, we would recommend increasing the number of colonies screened to account for the expected increased diversity of allele editing combinations

I will now outline the assay process using an essential gene as an example

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Target Essentiality Assay | Part 1 - Restriction Digest AnalysisSelection of a suitable restriction enzyme

• Target site of the guide RNA assessed for restriction endonuclease sites

• The ideal digest site spans the Cas9 cut site

TGGGAAATAGAAGCCAGTGAAGTGATGCTGTCCACTCGGATTGGGTCAGGCTCTTTTGGAACTGTTTAT

• Cells are infected with sgRNA in pLentiCRISPR (Sanjana et al, 2015)

• Cells are cultured in the presence of Puromycin for 2-3 weeks prior to plating for single cell colonies

• The relevant region is amplified and analysed by restriction digest

• Editing is detected by loss of the restriction digest site

• Both heterozygous and homozygous editing can be detected

sgRNA_Target

Hpy188 PAM

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Target Essentiality Assay | Part 1 - Restriction Digest AnalysisModelled data

• Disruption of the gene results in loss of the restriction enzyme site.

• The method can only determine whether disruption has occurred, not whether it is a frameshift.

All Alleles disruptedSome Alleles disruptedNo Alleles disrupted

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Target Essentiality Assay | Part 1 – Restriction Digest AnalysisExample results in a gene-target-dependent and -independent cell line

Cell line A – Independent of Target Cell line B – Dependent on Target

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Target Essentiality Assay | Part 2 – Fragment Analysis

• Selected colonies are analysed by fragment analysis

PCR

Fragment analyser

Fragment size In-del

Sample Name Allele 1 Allele 2 Allele 1 Allele 2

F12_Parental 417 0 A01_1D1 387 -30 G01_4H6 413 415 -4 -2 B03_2I9 402 417 -15 0 D04_3A12 422 5 B05_1N13 418 1 B01_4E1 416 417 -1 0 D02_5C7 414 417 -3 0 C03_3F9 418 1 E02_1F8 334 414 -83 -3 E03_4D9 369 417 -48 0 F04_4D12 413 -4 E05_2L13 374 -43 D01_3N2 408 417 -9 0 F02_2C8 426 9 F03_4G9 301 -116 F05_3B13 414 -3 B02_2E7 399 -18 G02_2L8 416 417 -1 0 B04_4J11 400 -17 G04_5I12 414 417 -3 0 G05_5E13 405 407 -12 -10 E01_1D3 369 -48 C04_5J11 417 423 0 6 H05_5O13 417 0 C06_4C17 414 418 -3 1 F01_3H3 390 -27 C02_4B7 413 -4

Non-disrupted Allele

Disrupted Allele

Expected size – 417bp

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Target Essentiality Assay | Part 2 – Fragment Analysis

• Results are correlated with restriction digest results to identify the true editing events in the clones

Homozygous editing confirmed by fragment analysis.

Homozygous editing revealed by fragment analysis.

Heterozygous editing confirmed by fragment analysis, presence of WT length fragment.

Heterozygous for fragment length, WT length fragment present.

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Target Essentiality Assay | Optional – Sequencing• If further confirmation of the editing events is required we can perform Sanger sequencing

• This can be used to verify the fidelity of the fragment analysis assay

Restriction digest assayFragment

length assay

Deletion by Fragment

assaySequencing data

Parental cells No disruption of FauI site 394 0bp …TTTGTGGTGGATGCAACCCGCAAGGGTAACAA… Wild type sequence

Example clone A

Homozygous disruption of FauI site

394&

3790bp & 15bp

…TTTGTGGTGGATGCAACCCGGAAGGGTAACAA… 1 bp substitution in FauI site

…TTTGTGGTGGATG----------------CAA… 16 bp deletion

Example clone B

Homozygous disruption of FauI site

394&

3470bp & 47bp

…TTTGTGGTGGATGCAACCTGCAAGGGTAACAA… 1 bp substitution in FauI site

…TTTGTGGT------------------------… 47 bp deletion

Example clone C

Homozygous disruption of FauI site

394&

3790bp & 15bp

…TTTGTGGTGGATGCAAAAAGCAAGGGTAACAA… 3 bp substitution

…TTAA----------------AAGGGTAACAA… 17 bp deletion + 2 bp substitution

Example clone D Homozygous disruption of

FauI site 394 0bp…TTTGTGGTGGATGCAACCCGAAAGGGTAACAA… 1 bp substitution in FauI site

…TTTGTGGTGGATGCAACCCGAAAGGGTAACAA… 1 bp substitution in FauI site

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Target Validation at Horizon | CRAF

CRAF was a target gene in Horizon’s Target Validation alliance with H3 Biomedicine Aim: Validate CRAF dependence in human KRAS mutant NSCLC – prediction of Barbacid and Tuveson papers

Cancer Discovery. 2011 Jul;1(2):128-36

Cancer Cell. 2011 May 17;19(5):652-63

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Target Validation at Horizon | CRAFApproach• 8 KRAS wild-type and 13 KRAS mutant NSCLC cell lines• Expression of dox-inducible shRNAs vs. KRAS (x3) , CRAF (x3) or controls in those lines• Assay CRAF dependence of growth in 2D – with follow up in spheroids, soft agar and in vivo experiments• Confirm knockdown and check for pathway modulation• Rescue phenotypes with shRNA resistant cDNAs & KI mutations

100ng/mlDox

WB: CRAF

WB: β-Actin

+ − + − + − + − + −

CRAF

#1

CRAF

#2

CRAF

#3

Scra

mbl

ed

Uni

nfec

ted KRAS WT NCI-H1299 cells

KRASWT cells were generally resistant to shRNA vs CRAF

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Target Validation at Horizon | CRAF

• shRNA results investigating CRAF dependence in KRAS mutant cells were ambiguous

• Good knockdown of the target was achieved, but only 2 of the shRNA produced an anti-proliferative phenotype

+− +− +− +− +−

CRA

F#1

CRA

F#78

4

CRA

F#91

1

Scra

mbl

ed

Uni

nfec

ted

100ng/mlDox

WB: CRAF

WB: β-Actin

50%

25%

KRASMT A549 cells

Overall KRAS mutant cells tended to be sensitive to shRNA vs CRAF, But effective CRAF KD can occur without growth inhibition (CRAF#784)cDNA rescue experiments were also inconclusive.

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Target Essentiality Assay | CRAF

We attempted to validate CRAF using an array-based sgRNA method, but this also resulted in ambiguous results .

An anti-proliferative phenotype was observed but this was not complete.

The Pathfinder Essentiality Assay, revealed CRAF was dispensable:

~30% of A549/sgCRAF clones have out-of-frame or large deletions in all 3 alleles of CRAF

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Target Essentiality Assay | WorkflowThis offering consists of a basic workflow, with optional “add-ons”; so you can tailor the work-plan to fit your needs.

Guide RNA evaluation (5xsgRNA)

Cell line evaluation (6 lines)

Main workflow

Optional “add-ons”*

Fragment analysis (48 samples per cell line

per target)

Additional fragment analysis

(per 48 samples)

Restriction digest analysis

(384 samples per cell line per target)

Additional restriction digest analysis (per 384 samples)

Cell line infection, growth and plate out (4

cell lines and 5x384-well plates per target)

Plate out of additional plates

(per 5x384 well plates)

Sanger Sequencing

* May incur additional costs

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Cell lines Available for Target Essentiality Assay (53 cell lines)

*Other cell lines can be evaluated for suitability with an extra fee

Tissue

Bone marrow K-562 KG-1

Breast MCF7 MCF10A MDA-MB-231 BT-474 BT-549 CAL-148 T47D

Colon/cecumDLD-1 HCT116 SW48 COLO 320DM LoVo LS411N RKO

VACO 432 SW480 HT29 HT55

Kidney 786-O RCC-MF G401

Lung A549 HCC827 NCI-H460 NCI-H520 NCI-H838 SK-LU-1

Oesophagus KYSE-70 KYSE-150 KYSE-270 TE-4

Ovarian A2780 CHO K1 ES-2

Pancreatic PANC-1 MIA-Paca-2

Prostate PC-3 DU145 LNCaP

Peripheral blood JURKAT NALM-6 RPMI-8226

Skin A375 IGR-1

Soft Tissue KYM-1 RD

Urinary bladder HT 1376 J82

Uterus/Cervix HEC-1-A HeLa SK-UT-1

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CRISPR-Cas9 | Functional Genomic Screening

• Functional genomic screening in pooled lentiviral system• Infrastructure adopted from shRNA screening• sgRNA enrichment scored by next generation sequencing

Mixed population Resistant cells SequencesgRNA Library

Library AssemblyLibrary Design sgRNA Library

Compound

Treatment

Cas9 + sgRNA

transduction

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CRISPR-Cas9 | CRISPRi and CRISPRa

• Repurposed screening using CRISPR technology• Catalytically-inactivated Cas9 (dCas9) fused to transactivation domain (CRISPRa for activation) or

repressor domain (CRISPRi for inhibition)• Target to gene promoters using sgRNAs• CRISPRi: Tackle essential genes• CRISPRa: Screen for gain-of-function mutations

CRISPRa West Coast CRISPRa East CoastCRISPRi

dCas9Catalyticallyinactive

dCas9-KRABfusion

Version 1 (2013)

Version 2 (2014)

Qi et al 2013

Gilbert et al 2013Gilbert et al 2014

Konermann et al 2015Mali et al 2014Gilbert et al 2013

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RepositioningPatient stratificationLOH2LTarget ValidationTarget ID

Genetic screens

Gene trapCRISPR-Cas9

siRNA

Knockdown or KO

Activity dead KI mutations

Generation of isogenic cell lines

MOA cell-based assays

Compound profiling in isogenic and non-isogenic cell panels

Working with Horizon | Services Available

Drug combination screening

Genetic screens

Target essentiality assay

TIDVAL alliances Target validation & early stage drug discovery collaborations

Finding a development path for stranded clinical assets

In vivo models including PDX models

Your Horizon Contact:

t + 44 (0)1223 655580f + 44 (0)1223 655581e info@horizondiscovery.comw www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Julie WickendenTeam Leaderj.wickenden@horizondiscovery.com+44 1223 655580 (ext 5664)