Faculty advisor: David LargaespadaThis research was supported by the Undergraduate Research Opportunities Program (UROP)

Dunnell, Justin

Knudson, Puggy

Kurata, Morito

Largaespada, David

Osborne, John

Peterson, Joe

Shafer, Paul

Slipek, Nicholas

Temiz, Nuri

Varshney, Jyotika

University of Minnesota Digital Conservancy

Validation of Candidate Drivers of Osteosarcomagenesis
with High-Throughput In Vitro Screening
Justin Dunnell1,3, Nicholas Slipek1, Jyotika Varshney1,2, Nuri Temiz1, Paul Shafer1, John Osborne1,3, Joe Peterson1,3, Puggy Knudson1,3, Morito Kurata1, David Largaespada1,4
1Masonic Cancer Center, Minneapolis, Minnesota;  2College of Veterinary Medicine, 3College of Biological Sciences, 4Department of Pediatrics, University of Minnesota
Background
Design
Results
- Sequence whole organ tissue from mice tail
vein injected with GeCKO transduced cell
populations for analysis of micrometastasic
clones
- Generate secondary guides against top gene
hits using Synthego chemically modified guide
RNAs
- Validate transformed phenotype of individual
gene knockouts in 2 additional OS cell lines
both in vitro and in vivo
- Perform CRISPRa screen for proto-oncogenes
in OS using newly validated library
Figure 1: Sequencing of integrated gRNA in early and end time point tumors. Sequencing of the transduced cell populations collected at the early time point (when the tumor
first became palpable), and endpoint (180 days). Three separate library transductions were preformed and 30E6 cells were injected into four mice each. Top ten gRNAs as a percent
of total reads for that sample are shown.
Osteosarcomas (OS) are rare, aggressive bone
cancers with a peak prevalence in late adolescence.
OS, particularly metastatic disease, has a very poor
prognosis and progress in developing new therapies
has been slow due, in part, to a paucity of
information on targetable genomic events
responsible for its development. Isolating genetic
mutations that drive the development and
progression of OS is complicated by the genomic
heterogeneity of the disease, which is almost
universally characterized by chromothripsis and
genomic instability. Previously, our lab has
completed a forward genetic screen for drivers of
osteosarcomagenesis in mice using an inducible
Sleeping Beauty system. To evaluate these drivers
and identify new ones, we chose to conduct a
CRISPR/Cas9 screen in a human cell line. Using a
CRISPR library that can target the entire genome
allows us to select for the development of specific
aggressive cancer phenotypes such as metastasis
and treatment resistance.
Future Work
Funding
1) Zach Sobiech Osteosarcoma Fund
2) Children’s Cancer Research Fund
3) American Cancer Society
Early Tumors Late Tumors
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28 days
43 days
60 days
A B
We chose a poorly metastatic, p53 wildtype, OS
cell line (U2OS) for these studies. A previously
generated lentiviral CRISPR library representing ~3x
genome coverage was used at a low multiplicity of
infection to ensure a single gRNA per cell. The
constitutive expression of the CRISPR/Cas9 system
will generate a diverse population of transform cells,
each carrying a unique mutation. When these cells
are subsequently xenographed into either the flank
or tail vein of NRG mice, they were exposed to a
tumorigenic bottle neck and cells where critical TSGs
had been disrupted should experience selective
outgrowth to form a tumor mass.
Next Generation 
Illumina Sequencing
Subcutaneous transplant into NSG mice
~6 weeks 26 weeks
Cas9-U2OS U2OS Transformed Cell Population
+ Cas9 
lentivirus
+ lentiviral 
sgRNA library
Liver Lung
Figure 2: Characterization of top screen hits. (A) gRNA abundance as a percent of total reads from all late time point tumors (n=9) and (B) top gene hits mapped according to
the the total number of late tumors they were identified in and the total percent of any tumor that gRNA comprised.
Conclusions
-There is a high variability in integrated gRNA
between early and late tumors
-Transduction replicates accounted for high
variability between early and late tumors,
verifying the validity of top gRNA targets
-A subcutaneous model of tumor growth
produces a strong selective pressure on edited
cell populations


Validation of Candidate Drivers of Osteosarcomagenesis  with High-Throughput In Vitro Screening

https://conservancy.umn.edu/bitstream/handle/11299/196269/Justin%20Dunnell%20Final%20Poster.pdf?sequence=1&isAllowed=y

Validation of Candidate Drivers of Osteosarcomagenesis with High-Throughput In Vitro Screening

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