how crispr–cas9 screening will revolutionise your drug development programs

HORIZON DISCOVERY

Benedict Cross | Team Leader, Discovery ScreeningWebinar October 21st 2015

Functional Genomic Screening with CRISPR-Cas9

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Human Healthcare Outcomes Defined by Patient GeneticsThe challenge has shifted from obtaining genomic information to understanding what it means

A missing link in correlating genetic variation with functional disease outcomes has been the availability of tools that enable us to edit the code of human cells with single base resolution and high-precision

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Products & Services at Horizon

Functional Genomic Screening

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Functional Genomics & the CRISPR Revolution

How Does CRISPR-Cas9 Screening Work?

Latest Developments – How to Push the Boundaries

Where Next?

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What Is Functional Genomic Screening for?

Genetic screening can be used to power discovery in myriad biological paradigms

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Horizon’s Functional Genomic Screening Portfolio

• Pooled screening with NGS read-out• Custom libraries to genome-wide• Robust, powerful & penetrant

CRISPR-Cas9

• Haploid cell knock-out screening• Genome-wide• High statistical power

Gene Trap Screening

• Arrayed screening• 2200 druggable genome library • Rapid, flexible & established

siRNA Screening

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Target ID | The Limitations of RNAi

Loss of function analysis using RNAi is inexpensive and widely applicable

However

Problems with RNAi can result in false positives or negatives

Only partial knockdown

Incomplete knockdown

Lack of reproducibility

Off-target effects

Little correlation between screens

HIV Host Factors

König et al. Cell

213 genesZhou et al.

Cell Host Microbe

300 genes

Brass et al.Science

273 genesTotal overlap only 3 genes

Off-target effects

Shalem et al Science 2014 Singh et al PLoS One 2015

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The CRISPR-Cas9 Gene Editing Platform

Pooled screening using CRISPR takes advantage of the nuclease activity of the Cas9 protein targeted to a precise genomic locus by a short guide sequence (sgRNA)

Cas9 endonucleasesgRNA

Target Genomic Locus

PAM siteTarget sequence

Site-specific dsDNA break

NHEJ and InDel editing

GENE KNOCKOUT

Anticipated to provide fewer off-target

concerns than RNAi

Robust phenotypes due to complete loss

of gene function

Cas9 enacts knock-out of target gene

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CRISPR-Cas9 Screening | Timeline of Development

2013

2014

2015

Present

Pre-publication of first full screening studiesShalem et al. 2014 & Wang et al 2014

Horizon launches GenASSIST CRISPR Programme

Horizon initiates CRISPR-Cas9 Screening in Cambridge, UK

Horizon completes first cell line engineering programme with CRISPR

Publication of seminal CRISPR-Cas9 studies in mammalian cellsJinek et al. 2012, Gasiunas et al 2012, Mali et al. 2013

First library (TSG) synthesis and assembly passes QC

First screening with small library data set (TSG)

Optimisation of screening programme for whole-genome screeningDefinition of bioinformatics pipeline for guide mapping and screen analysis

First client project completes with full data deliveryFirst whole-genome datasets in sensitivity and resistance screening

Launch of CRISPR-Cas9 Screening as research service

Optimisation of screening and initiation of over ten internal R&D screens

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Functional Genomics & the CRISPR Revolution

How Does CRISPR-Cas9 Screening Work?

Latest Developments – How to Push the Boundaries

Where Next?

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How Do CRISPR-Cas9 Screens Work?

Selection of genes to target anddesign of a suitable sgRNA library

Cell line is optimised and transduced with a pooled lentivirus library

Selected transduced cells are treated with the assay conditions

Deep sequencing is used to determine the abundance of each KO genotype

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Next Generation Sequencing for Pooled Screen Readout

Lentivirus library Control sample Test Sample

Deep sequencing to quantitatively identify the genotype of the cells in each sample

Use sequence of sgRNA as barcode for genotype

lentiviral expression cassette

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Positive and Negative Selection Screening

Genes which confer a growth advantage

in the assay conditions

Resistance screening

Genes which confer a growth inhibition

in the assay conditions

Sensitivity screening

Screen Optimised cell line

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Genetic Interaction Screening

Genotype X

Wildtype

Genotype Y

Mutant

Identify genotype-selective phenotypes

(e.g. synthetic lethality)

Can be conducted on panels of cell lines for

maximum target ID power

Cell divisions

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Example Screen: Resistance Screening

Recapitulation of existing screens using new library identified all six previously identified hits and several additional targets

A375 Transduce Cells(sgRNA Library)

BaselineSample

Control sampleDMSO

VemurafenibDrug treated sample

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Example Screen: Sensitivity Screening

Screening in haploid cells identified multiple mitochondrial complex I componentsStress-induced tumour suppressors TSC1/TSC2 also scored highly

NDUF family

members

TSC1 & TSC2

Gene ID

Novel hit

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Functional Genomics & the CRISPR Revolution

How Does CRISPR-Cas9 Screening Work?

Latest Developments – How to Push the Boundaries

Where Next?

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Optimisation of CRISPR-Cas9 KO Screening

Key questions in CRISPR-Cas9 Screening

What is the expected magnitude of drop-out for an essential gene with CRISPR-Cas9?

Can we improve this?

Library complexity and design: how many guides do we need and how do we design them?

How reproducible is a CRISPR-Cas9 screen?

What are the kinetics of CRISPR-Cas9 and how does this impact screen design?

Sequence-specific effects (with Desktop Genetics) – data in the pipeline

Use essential genes to monitor CRISPR-Cas9 activity

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Horizon’s New CRISPR-Cas9 Screening System

Evaluation of system performance using essential vs. non-essential gene depletionRobust overall mean drop-out in all collections of essential genes Significant improvement of screen performance with Horizon’s new optimised system

Essential gene collections Negative control gene collections

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pLentiCRISPRv2HD System

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Horizon’s New CRISPR-Cas9 Screening System

Guide-level drop-out analysis from guides targeting essential collection of genesDrop-out by up to 2000-fold detected using new HD vector systemHigh sensitivity even in challenging experimental paradigm (drop-out screening)

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Individual guides targeting essential genes

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Library Design for CRISPR-Cas9 KO Screening

Multiple competing guide design algorithms – which is best?Machine learned set (Wang et al. 2014) appears to show best performance overall. However, in Horizon’s improved system, both guide sets performed equivalently indicating the major determinant is the vector

Guides targeting essential genes

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GeCKOv2 Library Guides in pLentiCRISPRv2 GeCKOv2 Library Guides in Horizon vector

Wang et al. Library Guides in pLentiCRISPRv2 Wang et al. Library Guides in Horizon vector

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Reproducibility of CRISPR-Cas9 KO in Large Screens

Parallel analysis of guide performance in two cell linesData extremely well correlated data across whole library between cell lines – excellent reproducibility

Opens up fascinating opportunity for synthetic lethal target discovery – real targets should be robustly identified

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A375 cells (triploid)eHAP cells (Haploid)

Individual guides targeting essential and non-essential genes

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Kinetics of CRISPR-Cas9 KO & Design Considerations

Analysis of time-resolved samples of guides targeting essential genesIn our new backbone, the loss of essential genes can occur very rapidly

Particularly important for hit calling in genetic interaction studies where longitudinal samples are crucial

Sample collection pointPlasmid input T1 T2 T3 T4

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Functional Genomics & the CRISPR- Revolution

How Does CRISPR-Cas9 Screening Work?

Latest Developments – How to Push the Boundaries

Where Next?

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Forward Genetic Screening with Horizon Reporter Cell Lines

Horizon Engineered Reporter Cell line

Transduced cellsCas9/sgRNA

Deep Sequencing for Target ID

GFP fluorescence

Assay condition

Flow cytometry to sort responsive populations

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6-TG resistance screen in eHAP cells shows MMR genes as top hits

Validation of MSH6 and MLH1 with off-the-shelf haploid KO cells (96 h assay)

Target Validation Using Horizon KO Cell Lines

Whole genome screen Prosecco plot Validation of top hits with engineered KO cell lines

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CRISPR-dCas9 Transcriptional Regulation Screening

• Repurposed screening using CRISPR technology• Catalytically-inactivated Cas9 (endonuclease-dead) fused to transactivation domain • Target dCas9 to gene promoters using sgRNAs• CRISPRi: Tackle essential genes• CRISPRa: Screen for gain-of-function mutations

Konermann et al 2015Mali et al 2014

Gilbert et al 2013Gilbert et al 2014

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Project phases

1. Library design and generation (optional)2. Cell line optimisation3. Screen initiation and sample collection4. Sample preparation and NGS5. Screen QC and analysis6. Hit nomination by Horizon scientists

Deliverables

A final report containing all raw & analysed data and hit nominations

Turnaround time

14-20 weeks

Horizon’s CRISPR-Screening Service

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CRISPR-Cas9 Screening - Join the Revolution!

Why work with Horizon?

• Horizon has a strong IP position with three licensed estates around CRISPR-Cas9

• Benefit from our library design expertise including robust control sets defined by us

• Access our proprietary backbone providing >ten-fold increase in performance

• A complete workflow, from design to hit nomination and excellent customer support

www.horizondiscovery.comresearchservices@horizondiscovery.com

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

Benedict Cross, PhD Team Leader | Discovery Screeningb.cross@horizondiscovery.com01223 655580