LIVE 9/19 8AM – 10AM USING CRISPR/Cas9 FOR FUNCTIONAL SCREENING
CHI’s 2nd Annual Symposium CRISPR: Mechanisms and Applications @CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston
http://www.discoveryontarget.com/
http://www.discoveryontarget.com/crispr-therapies/
Meeting #: #BostonDOT16
Meeting @: @BostonDOT
Leaders in Pharmaceutical Business Intelligence (LPBI) Group
is a Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.
In Attendance, streaming LIVE using Social Media
Aviva Lev-Ari, PhD, RN
Editor-in-Chief
http://pharmaceuticalintelligence.com
Monday, September 19
7:00 am Registration Open and Morning Coffee
COMMENTS BY Stephen J Williams, PhD
8:10 Comparing Arrayed siRNA and CRISPR Approaches Towards Functional Genomics Screening
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.
- Array screening: microplate screens: genome wide pooled screens take a long time under gold standard; pooled screens use alot of cell culture
- Enrichment screens many assays are not compatible with pooled screens; assays that look at cytokines released from cells so need an arrayed format
- Off target effect dominate sirna screens: hope 2 sirna target the same thing so most of what we see is off target
- The CRSPRCas9 system: indels to change function
- Use pooled gRNA plasmid libraries and use lentiviral infections, pcr amplification of gRNAs, use NGS to verify and sequence
- Negative selection screens are more noisy than positive selection screens eg. a hairpin screens would give you offtarget (50 more than CRSPR
- Issues with lentivirus: doing a large screen and upscaling 1) safety 2) reproduceability of infection 3) can take 7 to 10 days to see effect in small 96 well
- Dharmacon has synthetic cRNAs so basically you need cells with Cas9 and precomplexing crRNA and tacrRNA prior to transfection in cell (reverse transfection)
- Can use this strategy for cell cycle proteins
- crRNAs are dependent on Cas9 so little off target effect: however a portion of cells that are resistant to editing (polyclonal population of Cas9 cells: **** SO YOU MUST pick clones
- Then you will see 80% indels: good editing
- NOT all crRNAs are equal: see heterogenous response
- When do a larger screen get more hits with crRNA than siRNA
- You can use CRSPR also to verify the hits by introduce the gene by CRSPR into same cells that have Cas9 and recapitulate phenotype (good control)
- QuESTION: does picking Cas9 clones alter the phenotype of original cell source?
- They did not see their target phenotype later (are there false negatives hidden in the screen?); can we identify workhorse systems where this works well? MANY crRNAs are NOT EFFECTIVE: risk of high false negatives
QUESTION: Are you getting better editing? Use the positive control genes to establish system, refine CRSPR algorithms
QUESTION: some of the guides are targeting isoforms that don’t exist in the cells so that is why you don’t get high % of hits (1:4, 1: 8). Some toxicity with tracer RNAs (interferon response)
TALK SPECIFIC # and @
#Crisper
#CRSPR #Cas9 #geneediting #genomics |
@genentech |
8:40 Getting from Alpha to Omega: Successfully Conceptualizing, Starting and Finishing CRISPR/Cas Screens
Ralph Garippa, Ph.D., Director, RNAi & Gene Editing Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center
Collaborated with Danwei Huangfu, PhD: generates iPSC lines particular beta cell in pancreas
- Using HDAC inhibitors and demethylase inhibitors valproic acid increases efficiencies of reprogramming
- Biocyte with Tacra taking these iPSC cells did a deal with academia
- Viacyte 22015 putting the iPSC in a semipermeable membrane so don’t get immune and no glucose tolerance (lifespan 32 days in rodents), deep in phase 1 and 2
- Have to use WNT and activin A to bring to endoderm ; each lineage requires its own factors
- EOMES is necessary but not fully sufficient for differentiation
- So generated a iCas9 SOX17 (gives GFP signal off to on)
- Focused on OCT4 and NANOG from Activin (TGFB dependent)
- So with screens you are always looking for the outliers away from controls and rank and file of library (use the z score best more than a log fold outlier e.g. log2)
- Problem: if doing these screens need to get IP from Genentech
- Linkes Gecko version 2 over one for crgRNA libraries; avialable at Broad but must do NGS to verify library
TALK SPECIFIC # and @
#Crisper
#CRSPR #Cas9 #geneediting #genomics #diabetes |
@CHI
@sloan_kettering @MSKCC_OncoNotes @Boston @BiotechNews @pharma_BI @AVIVA_1950 |
9:10 Genome Editing-Enabled HTS Assays for Genetically Inherited Disease Drug Discovery
James Inglese, Ph.D., Head Assay Development & Screening Technologies, National Center for Advancing Translational Sciences, NIH
- GAN giant axonal neuropathy loss of gene gigaxon (involved in protein clearance build up proteins (fibrils) in neuronal axons)
- Most HTS looked at promoter regions but now with CRSPR can make more physiologically relevant
- Screening insoluble compounds: might be potent but insoluble (potent but flat DR) and chemists could make more soluble form
- Using a intronic enhancer in promoter region could get random integration and strong transcriptional repression in a chemical screen (problems: small sequence of regulation so small section of gene regulatory elements, would not be susceptible to miRNA regulation, context epigenetic responses)
- Genome editing can help solve these problems
- Use two reporters (orthogonality); important to have two target pathways
- These orthogonal assays pulled out a very effective compound which not seen in single reporter assay
- LOSS of ACTION ASSAY: looking for modulators, loss vs gain of signal finding molecules that have off target usually; looking for the release of inhibitors of your target (could do this for transcriptionally repressed genes?); do this by generating a bicistronic reporter (coincidence reporter cell lines)
- They did this with parkin gene and put in a coincidence reporter and allows the detection of concordant activities (determine if artifacts are in fact good signals) then run the HTS
- Genome editing allowed for detection of new pathways not detectable with previous promoter based strategy
TALK SPECIFIC # and @
#geneediting
#raredisease #genetics #AssayDevelopment #NIH #CMT #drugdiscovery |
@BostonDOT
@NIH @BiotechNews @PharmaNews @pharma_BI @AVIVA_1950 @DrugDiscover365 |
9:40 Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.
They are interested in finding VHL targets using genome wide CRSPR KO screen for renal cancer *VHL also involved in hypoxic response but look at our other posts on other factors effecting VHL hypoxic response including AMPK
- So for isogenic cell lines if Cas9 active KO if cas9 not active no VHL KO; so can use an exogenous delivery of Cas9, the talks previously used cells that already contain Cas9
- MAGeCK software enables robust identification of essential genes from genome scale CRSPR KO screens (2014 Nature Biology)
- Used a bait screen to screen for immuno-suppressive molecules
- Replicates don’t reflect what is in the library high signal to noise; should run orthogonal screens
- Inducible CRSPR: cas9 under tet inducible promoter
- Can get around problems with in vivo by doing an ex vivo strategy
- For primary cells it is work in progress (multiple issues including lentiviral transfection of cas9
TALK SPECIFIC # and @
#geneediting
#CRISPR #Cas9 #NIH #CMT #drugdiscovery #pharmanews #CRSPR #genomics |
@BostonDOT
@abbvie @BiotechNews @PharmaNews @pharma_BI @AVIVA_1950 @DrugDiscover365 |
USING CRISPR/Cas9 FOR FUNCTIONAL SCREENING
8:00 Chairperson’s Opening Remarks
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.
- Off target effects emerged and interpretation difficulties emerged as well.
- CRISPR – and screening and how using the Screening technology for experiments
8:10 Comparing Arrayed siRNA and CRISPR Approaches Towards Functional Genomics Screening
Scott Martin, Ph.D., Group Lead, Functional Genomics, Genentech Inc.
RNAi has been a workhorse for loss-of-function screening. Although powerful, RNAi is hampered by false positives. New screening technologies based on CRISPR/Cas9 appear less prone to off-target effects. CRISPR/Cas9 screens are conducted in pooled formats. However, this format is not amenable to many assays. In an effort to expand its utility, we explored the use of arrayed CRISPR/Cas9 screening with synthetic CRISPR RNAs.
Arrayed Screens vs Genome-wide pooled screens for proliferation-type assays. Many assays are not compatible with pooled screening Like: Cytokines, assays need remain unpooled.
- Off target effects Dominate siRNA Screens
- CRISPR-Cas9 Nuclease, crRNA tracrRNA
- Edit genes for Pooled gRNA plasmid libraries
- positive selection screen of a few thousand genes reveals very clean screen – 50 non-targeting controls show very little activity
- Arrayed CRISPR: Lenti delivery of gsRNA to Cas9 expressing cells can induce penetrant phenotypes in plate-based format.
- Evaluating Synthetic and Tracer RNA: crRNA (CRISPR RNA) – an Alternative to siRNAs (tracrRNA)– scalable abd automatable with CRSPR reagents
- DNA replication – the knockdown of certain proteins: COntrols agains geminin (GMNN) – maximum phynotype at transfuction
- Synthetic crRNA Effects are dependent on CRISPR Cas9
- More expressing clone expess more Cas9
- loss of protein in GMNN editing clone
- Nor all crRNAs are equal: Heterogenous response
- Screening with Synthetic crRNA
- targeting 650 ubiquitin-related genes with 4 crRNAs per gene
- siRNA: DIfferent crRNA targeting the same gene exhibiting more correlation than siRNA designed to target the same gene.
- better correlation guides targeting same gene
- top crRNA Screen Candidates – more responsive
- GO Term ENrichment
- Testing Edit-R with a Different System -1
- Novel candidate genes
- known control genes
- Immunofluorescence assays: DNA replication controls
- comparison of siRNAs vs crRNAs
Key question: is one active crRNA good enough if OTEs are not as big a factor? identify few workhorses systems that crRNA work well in like HAP1
Summary
- GMNN responsive in screening
- Sequence Scoring algorithms for targeting editing
- assay effective stability of the agent — phynotype is stable, clone population – several days
- Interferon response – toxicity with crRNA
- Pooled vs Arrays RNA – some labs do both some do only one of these
8:40 Getting from Alpha to Omega: Successfully Conceptualizing, Starting and Finishing CRISPR/Cas Screens
Ralph Garippa, Ph.D., Director, RNAi & Gene Editing Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center
For certain molecular targets, to unravel the underlying biology of loss-of-function studies, it is simply not enough to potently knockdown the protein. In some cases, a complete functional knockout is called for. Here we summarize our early experiences, highlighting the strengths of this new powerful system but also calling attention to technical areas which need to be addressed and further improved as the technology moves deeper into the mainstream.
- Custom individual Crispr construct:
- cut, nick
- custome pooled libraries
- select lentiviral vectors
- Off-the-shelf Library Expansion and QC: Human and Mouse
- Genomic DNA: CRISPR
- human pluripotent stem cells hPSC
- Forward Genetics vs Reverse Genetics
- Endodermal: all the GI – Beta cells in Pancereas
- Viacyte.com – create cells for implantation, Beta cells in Pancreas – after 75 implantation balabced glycemia in rats
- Markers; Wnt, ActA
- sgRNA vs shRNA – pooled screens
- Endoderm differntiation relies on TGF betaand WNT Activation
- Positive control: EOMES – mouse embryo
- Whole-genome Library Infection >> Mutagenesis >> Differentiation – % of Total cells Infected 30%
- SMAD2 SOX17 – Endodermal differentiation
- Hit validation – tested 22 od the top 50 positive regulators
- Known positive regulators in the tGFBeta Pathway
Conclutions
- Organogenesis regulation of — new understanding of
- Gene knockout strong phynotype identification
9:10 Genome Editing-Enabled HTS Assays for Genetically Inherited Disease Drug Discovery
James Inglese, Ph.D., Head Assay Development & Screening Technologies, National Center for Advancing Translational Sciences, National Institutes of Health
The targeted precision of genome editing was used in combination with advances in reporter gene design to modify the genetic loci of neurologic target genes to create HTS assays for compound library interrogation. Our goal was to identify transcriptionally active pharmacological agents acting by a variety of mechanisms, including through chromatin co-regulators accessible by our assay design. Specific case studies will serve to illustrate progress and findings to date.
- ADST Lab: Assay Development & Screening Technology Lab
- Disease Foundation
- Postdocs working on NCATS
- GAN – Giant Axonal Neuropathy: Gigaxonin – gene responsible for filaments causing disease – dementing cells as a result of gene dysfunction
- Gene Therapy: Clear aggregates from Exon: Flanking primers Exon Targeting region PAM
- Evaluation of chemical libraries with quantitative high throughput screening (qHTS)
- Structure Activity Relationship (SAR) – Robots – Automated large scale screening
- CMT – CHarcot-Marie-Tooth two copies of mylan sheet – pmp22 – Degenerative neuropaty – HTS assay development strategies for transcriptional PMP22 down-regulation – Swann cells in Rat of the Sciatic nerve
- Limitation:
- not sensitive to posttransctitional mechanisms microRNA
- context-dependent epigenetic regulation unrelated to naive
- Pharmacology of active compounds from PMP22 reporter assays
- Compound tested: Bryostatin
- Reporter Gee Assays: Loss-of-signal vs Gain-of-signal: Basal output va Treatment output: ENzyme assay vs Cell assay
- SOlving compound-mediated reporter t1/2 effects with coincedence detection
- Ribosome “Skipping”
- CHemical biology and Drug discovery to install CR parkin in Parkinson Disease
- Activity signature from a chemical ibrary qHIS
- New MOA which regulate
- Recommendations for reporter gene assays
- New genome editing
- Targeted phyenotypic assays designed using geneome editied
9:40 Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.
Advances in CRISPR gene editing and genomics technologies are rapidly changing biopharmaceutical R&D landscape, from target ID and validation, to drug mechanism of action, and to translational science. We will use vignettes of various genomics research applications within AbbVie R&D environment as a witness to this paradigm shift currently ongoing in the drug industry.
Data fro Cook 2014 NRDD
- right biology .. right target.. right molecule .. right dose .. right patient
- Functional Genomics: CRISPR
I. Gene pertubation libraries allow functional analysis of all genes
- Two types of NGS application
- Screening design for VHL-synthetic lethal KO
- Construction of VHL isogenic pair cell line: VHL-mutant cell lines (786-O, A-498)
- TCGA 2013 Nature
- Genome-wide CRISPR KO screen to identify novel VHL synthetic lethal targets
- Pooled lentiviral screening based cellualr barcoding: Pooled Infection and Screening Assays
- NA Seq
- Pooled screening deconvolution
- VHL CRISPR KO screening hits: mTOR inhibitors, temsirolimus, everolimus – 1st and 2nd line therapies – VHL protects cells from essential gene KO
- Receptor ID screen for an orphan immunosupressive ligand – receptor for ligand not known – detect aAb –>> IgG1-APC
- ligand binding – FACS sorting and NGS – Normalizeation of hits against noise – critical for eliminating bias present in screening library
- CRISPR/CRISPRi vs CRISPR KO: Transcription Activation & Supression
- Inducible CRISPR KO system
II. In vivo Immuno-oncology screening: Tumor target and immuno targets
III. Challenges in Primary Human cells
functional genomic analysis of human genetic variants based on CRISPR KO and KI larger role in new target discovery in the post-genome era
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