how crispr–cas9 screening will revolutionise your drug development programs
TRANSCRIPT
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
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
Benedict Cross, PhD Team Leader | Discovery [email protected] 655580