reference standards, gdna, ffpe (mapk, braf, egfr, kras)
TRANSCRIPT
ONCOREF™ Molecular Reference Standards: Application of CRISPR/Cas9 to the Generation of Footprint-Free
Isogenic Cell Lines and Nucleic Acid Reference Materials
Advancing Genome Engineering
Andrew Hilmer, Ph.D.Product Manager
MAPK Mutation SeriesOctober 4, 2016
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• Background
• Isogenic Cell Line Generation
• Application to Molecular Reference Standards
• Other Custom Services at Applied StemCell
Outline
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• In 2016, in the US1:
– 1.7 M new cases of cancer diagnosed
– 600,000 people will die from the disease (~1 person/minute)
• 3-35% of premature deaths could be avoided with better screening methods2
Why Did We Develop this Product Series
Goal: Develop a toolset that can be used for enabling research into better diagnostics and treatment
1. Am Canc Soc. “Cancer Facts and Figures 2016”2. www.cancer.gov
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• 51 Cell Lines Engineered Using CRISPR/Cas9
– MAPK mutations from COSMIC
– 32 Mutations in EGFR (RKO)
– 12 Mutations in KRAS (HCT-116)
– 7 Mutations in BRAF (RKO)
• Isogenic Positive and Negative Controls
• Mutations are Homozygous and Footprint-Free
• Available in a Number of Formats:
– Genomic DNA
– FFPE Slides, and Scrolls
– Cell Lines
Introduction to ONCOREF™ Product Series
Research Use Only
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Background on MAPK Pathway
• Pathway involved in:
– Cell proliferation, cell death, differentiation, migration, and invasion
• Frequently mutated in cancer:
– KRAS mutations in >90% of pancreatic cancers1
– BRAF mutations in ~50% of melanomas2
– 20-50% of NSCLC are EGFR mutant3
1. Eser, S et al. Brit. J. Cancer. 111, p817-822 (2014)2. Ascierto, PA et al. J. Transl. Med., 10:85, (2012)3. Midha, A et al. Am. J. Canc. Res, 5, p2892-2911 (2015)
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MAPK Cell Line Series:Cas9-Mediated Genome Engineering
gRNA directs Cas9 to target site
DSB
No RepairTemplate Available
RepairTemplate Available
NHEJ HDR
Error-ProneOften Results in Indel Formation
PreciseUses the Template You Provide
1 2
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MAPK Cell Line Series:Cas9 for Footprint-Free Genome Editing
Footprint-FreeFootprint
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MAPK Cell Line Series:Cell Line Engineering Workflow
Clonally Homogeneous Genetically Validated
Homozygous Modification
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Single Cell Cloning
Clonal Expansion
Cas9 Transfection
Example 1: Point Mutation Example 2: Small Insertion Example 3: Deletionc.2310_2311insGGTp.D770_N771insG
c.2303G>Tp.S768I
c.2239_2256del18p.L747_S752del
MAPK Cell Line Series:Engineering Clinically Relevant Mutations
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*
*****
************
*******
***
*
*
= Exclusive to ASC *
12 KRAS Mutations
7 BRAF Mutations
32 EGFR Mutations
**
Research Use Only
MAPK Cell Line Series:Full Mutation Panel
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MAPK Cell Line Series Highlights:Largest Panel of EGFR Exon 19 Mutations
• 22 Deletion Mutations
• In-frame mutations that occur within kinase domain
• 21 mutations are unique to ASC
c. 2238_2252>GCAp. L747_T751>QCOSM12419
COSM12422c. 2238_2248>GCp. L747_A750>P
Exon 19 Mutation Panel
Cheng, L, et al. Modern Pathology, 25, p 347-369 (2012)
Research Use Only
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Assay Development
Optimize sample processing protocols
Establish limit of detection (LOD)
Identify and validate optimal reagents
Monitor the impact of workflow changes on results
Quality Control and Assay Reproducibility
Accurately evaluate batch-to-batch variability
Easily transfer workflows across labs or institutions
MAPK FFPE Cell Line Standards:Consistent Source of Bio-Relevant Specimens
Research Use Only
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Tumor Xenografts
Plasmids
Amplicons
Genomic DNA
FFPE Cell Blocks
Bio-RelevantHighly-Reproducible
Yes No
Yes
Yes
Yes
No
No Yes
Yes
Yes
Research Use Only
MAPK FFPE Cell Line Standards:Consistent Source of Bio-Relevant Specimens
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Real Mutation
Barcodes
Amplify and SequenceAttach Random Barcodes
= true mutations= PCR and/or sequencing errors
Form Consensus Sequences
Pre-processing Post-processingError Rate ~ 0.25% Error Rate: 0.009%
Molecular Reference Standards:Genomic DNA
Development of Library Prep and Data Processing Pipelines
Implementation of Methods for Improved Detection of Somatic Mutations
High purity, high molecular weight gDNA
Perfect starting point for assay development
Research Use Only
Spik
ed in
mu
tan
t D
NA
to
~0
.2%
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• Formalin-fixation provides a means of preserving biological specimens
• Great at preserving tissue morphology
• Allows for banking of tissues for analysis at later time points
Molecular Reference Standards:Overview of FFPE Samples
www.bethyl.comwww.biosb.com www.intellmed.eu
Research Use Only
FFPE Benefits
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• Often give low extraction yields for nucleic acids
• DNA is often significantly degraded
– dsDNA breaks resulting in fragmentation
– Cytosine deamination (C > U)
www.bethyl.comwww.biosb.com www.intellmed.eu
FFPE Challenges
Research Use Only
Molecular Reference Standards:Overview of FFPE Samples
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Molecular Reference Standards:FFPE Cell Line Blocks
Research Use Only
Your Applications
FFPE Scrolls
FFPE Slides
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Block 1 Block 2 Block 3
4x
20x
Molecular Reference Standards:FFPE Slides
Consistent and reproducible block generation
5 µm Sections, Hematoxylin Stained
Research Use Only
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Molecular Reference Standards:Intra-block Consistency
Section 1 Section 200 Section 400
• 5 µm sections
• Hematoxylin and eosin staining
• Approximately 500 sections per block
Research Use Only
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Molecular Reference Standards:FFPE Scrolls
BRAF V600E TaqMan Assay
Research Use Only
Qua
ntifluo
r
Nan
odro
p0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Extr
acte
d D
NA
(ng)
• 15-20 µm sections
• Simulate processing of biological specimens
• Perfect for optimizing DNA extraction or assay development
FFPE Scrolls
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Single Cell Cloning and Genotyping
Positive Clone Expansion
Cas9 Transfection
Project Step Timeline (3-5 months)
Custom Cell Lines and Reference Materials:CRISPR Cell Line Services
Research Use Only
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Histology Solutions:Custom FFPE Slide Services
FFPE Cell Blocks
Benefits• Use your own cell lines
• Consistent, reliable block generation
• Single- and multi-spotting services
SiHaHeLaOVCAR-8C-33ACa Ski
Research Use Only
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Summary:MAPK Molecular Reference Standards
• 51 Cell Lines Engineered Using CRISPR/Cas9
• Isogenic Positive and Negative Controls
• Available in a Number of Formats:
– Genomic DNA
– FFPE Blocks, Slides, and Scrolls
– Cell Lines
• Mutations are Homozygous and Footprint-Free
• Custom Cell Line and FFPE Services
