How CRISPR–Cas9 Screening will revolutionise your drug development programs
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CRISPR–Cas9 mediated genome editing provides a novel and highly efficient way to probe gene function. Using this technology, thousands of genes can be knocked out and their function assessed in a single experiment. This makes CRISPR–Cas9 screening a powerful tool for drug target ID and validation, understanding drug mechanisms of action and patient stratification. In this webinar, we use our experience with CRISPR–Cas9 to discuss the power and applicability of CRISPR-Cas9 screening technologies. We focus on how to use this technology to address important biological questions, and consider what’s possible, what’s plausible and what constitutes a ‘hit’. We also highlight Horizon’s latest developments to the CRISPR-Cas9 screening platform.
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How CRISPR–Cas9 Screening will revolutionise your drug development programs
- 1. HORIZON DISCOVERY Benedict Cross | Team Leader, Discovery Screening Webinar October 21st 2015 Functional Genomic Screening with CRISPR-Cas9
- 2. 2 Human Healthcare Outcomes Defined by Patient Genetics The 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
- 3. 3 Products & Services at Horizon Functional Genomic Screening
- 4. 4 Functional Genomics & the CRISPR Revolution How Does CRISPR-Cas9 Screening Work? Latest Developments – How to Push the Boundaries Where Next?
- 5. 5 What Is Functional Genomic Screening for? Genetic screening can be used to power discovery in myriad biological paradigms
- 6. 6 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
- 7. 7 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 genes Zhou et al. Cell Host Microbe 300 genes Brass et al. Science 273 genes Total overlap only 3 genes Off-target effects Shalem et al Science 2014 Singh et al PLoS One 2015
- 8. 8 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 endonuclease sgRNA 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
- 9. 9 CRISPR-Cas9 Screening | Timeline of Development 2013 2014 2015 Present Pre-publication of first full screening studies Shalem 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 cells Jinek 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 screening Definition of bioinformatics pipeline for guide mapping and screen analysis First client project completes with full data delivery First 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
- 10. 10 Functional Genomics & the CRISPR Revolution How Does CRISPR-Cas9 Screening Work? Latest Developments – How to Push the Boundaries Where Next?
- 11. 11 How Do CRISPR-Cas9 Screens Work? Selection of genes to target and design 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
- 12. 12 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
- 13. 13 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
- 14. 14 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
- 15. 15 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) Baseline Sample Control sample DMSO Vemurafenib Drug treated sample
- 16. 16 Example Screen: Sensitivity Screening Screening in haploid cells identified multiple mitochondrial complex I components Stress-induced tumour suppressors TSC1/TSC2 also scored highly NDUF family members TSC1 & TSC2 Gene ID Novel hit
- 17. 17 Functional Genomics & the CRISPR Revolution How Does CRISPR-Cas9 Screening Work? Latest Developments – How to Push the Boundaries Where Next?
- 18. 18 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
- 19. 19 Horizon’s New CRISPR-Cas9 Screening System Evaluation of system performance using essential vs. non-essential gene depletion Robust 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 Changeinabundanceovertime(LogFoldChange) pLentiCRISPRv2 HD System
- 20. 20 Horizon’s New CRISPR-Cas9 Screening System Guide-level drop-out analysis from guides targeting essential collection of genes Drop-out by up to 2000-fold detected using new HD vector system High sensitivity even in challenging experimental paradigm (drop-out screening) Changeinabundanceovertime(LogFoldChange) pLentiCRISPRv2 HD System Individual guides targeting essential genes
- 21. 21 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 Changeinabundanceovertime(LogFoldChange) 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
- 22. 22 Reproducibility of CRISPR-Cas9 KO in Large Screens Parallel analysis of guide performance in two cell lines Data 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 Changeinabundanceovertime(LogFoldChange) A375 cells (triploid) eHAP cells (Haploid) Individual guides targeting essential and non-essential genes
- 23. 23 Kinetics of CRISPR-Cas9 KO & Design Considerations Analysis of time-resolved samples of guides targeting essential genes In 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 point Plasmid input T1 T2 T3 T4 Normalisedabundance(Log2counts)
- 24. 24 Functional Genomics & the CRISPR- Revolution How Does CRISPR-Cas9 Screening Work? Latest Developments – How to Push the Boundaries Where Next?
- 25. 25 Forward Genetic Screening with Horizon Reporter Cell Lines Horizon Engineered Reporter Cell line Transduced cells Cas9/sgRNA Deep Sequencing for Target ID GFP fluorescence Assay condition Flow cytometry to sort responsive populations
- 26. 26 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
- 27. 27 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 2015 Mali et al 2014 Gilbert et al 2013 Gilbert et al 2014
- 28. 28 Project phases 1. Library design and generation (optional) 2. Cell line optimisation 3. Screen initiation and sample collection 4. Sample preparation and NGS 5. Screen QC and analysis 6. 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
- 29. 29 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.com researchservices@horizondiscovery.com
- 30. Your Horizon Contact: t + 44 (0)1223 655580 f + 44 (0)1223 655581 e info@horizondiscovery.com w www.horizondiscovery.com Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom Benedict Cross, PhD Team Leader | Discovery Screening b.cross@horizondiscovery.com 01223 655580
Editor's Notes
- Ben