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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 Series
October 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 Repair
Template Available
Repair
Template Available
NHEJ HDR
Error-Prone
Often Results in Indel Formation
Precise
Uses 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: Deletion
c.2310_2311insGGT
p.D770_N771insG
c.2303G>T
p.S768I
c.2239_2256del18
p.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
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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>GCA
p. L747_T751>QCOSM12419
COSM12422
c. 2238_2248>GC
p. 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-processing
Error 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
Spikedinmutant
DNAto~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
Q
uantifluorN
anodrop
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
ExtractedDNA(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

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Thank You!!
info@appliedstemcell.com
www.appliedstemcell.com