1) The authors are using the CRISPR-Cas9 system to induce double-strand breaks near centromeres on chromosomes 3p and 8p in order to generate models of partial aneuploidy through homologous recombination. 2) They have successfully targeted and induced breaks on these chromosomes, and selected for cells that underwent recombination to replace the chromosome arm with an artificial telomere. 3) In the future, they aim to characterize the phenotypic and tumorigenic effects of specific chromosomal arm losses to further understand their role in cancer formation and progression.

1. Genome engineering to generate models of chromosome arm-level aneuploidies
Alison M. Taylor1,2, Michael S. Cuoco1, and Matthew L. Meyerson1,2
1Dana-Farber Cancer Institute, Division of Medical Oncology 2Broad Institute Cancer Program and Harvard Medical School
Over 90% of tumors are aneuploid and have arm- or whole- chromosome level copy
number changes. Arm-level copy number alterations cluster by tumor type (such as loss of 3p
in lung or 8p in colon), suggesting that specific arm-level changes are influenced by cell type.
Aneuploidy is known to contribute to tumor development; however, studies show that
aneuploid cells with one extra chromosome actually have decreased fitness and growth.
Systematic methods of generating copy number changes on a specific chromosome have not
been tested, leaving the effects of specific aneuploidies in cancer unclear. Recent advances
in endonuclease technology have allowed increased efficiency in generating double-strand
breaks. We are using the CRISPR-Cas9 system to generate double-strand breaks (DSBs) to
induce copy number changes. The goal of this work is to generate partial aneuploidies of
arm-level loss. We have targeted guide RNAs adjacent to centromeric satellite-repeat
sequences on chromosome arms 3p and 8p. At these locations, we have successfully
induced homologous recombination with a selection cassette and artificial telomere. Initially,
we are optimizing the technique in 293T cells before moving to primary or immortalized cells,
where we will identify the downstream phenotypic and tumorigenic effects. These studies
address a gap in our understanding of aneuploidy in cancer by using targeted endonuclease
technology to create models of partial aneuploidies. Studies in these models will provide us
with knowledge of how different chromosomal changes contribute to cancer formation, and
have implications on our understanding of tumorigenesis.
Abstract
Figure 2. Selection of CRISPR target location adjacent to the centromere.
Sequence directly adjacent to the centromere is highly repetitive, so nuclease target regions
were selected just outside of this region.
Nuclease Target
Location
Centromere Pericentromeric
Region
Repetitive Sequence
CRISPR Number
1 2 1 2 ---
Figure 3. Surveyor assay demonstrates
Cas9 activity near the centromere of 3p.
Two CRISPR guides were tested in 293T cells (1 and
2). A cleavage product below the PCR band indicates
successful nuclease activity by the surveyor assay.
CRISPR #2 has generated double strand breaks within
this region.
Nuclease Target
Location
X
HR
PURO
HR
PURO
Telomere
Containing
Plasmid
Artificial
Telomere
+
Figure 4. Schematic of recombination based approach.
A plasmid containing 1kb of homologous DNA, a puromycin selection marker, and artificial
telomere is co-transfected with a CRISPR-Cas9 construct to target DNA sequence adjacent to
the centromere. Upon transfection, a double strand break is produced and repaired by
homologous directed recombination, removing a chromosome arm replacing it with an artificial
telomere. The remaining chromosome arm is likely degraded, but may bind to another
chromosome.
Unselected
Puro
Selected
1 2 -- 1 1 2 2
Recombination
Plasmid Alone
1500
1000
500
• The CRISPR-Cas9 system is able to generate double strand breaks near the centromere.
• Homologous recombination of an artificial telomere in place of a chromosome arm can be
selected for at the site of a targeted double strand break near the centromere.
• We are currently isolating clones with this recombination event, and will confirm that the 3p
or 8p arm is lost by FISH. In addition, we will generate the recombination in immortalized
lung and colon cells.
• Once we have successfully generated a model of chromosome arm-level loss, we can
continue with phenotypic characterization to determine its role in tumorigenesis. Future
studies include testing for effects on proliferation, invasiveness, and transformation
Summary and Future Directions
Funding Sources
Figure 1 (adapted from Hoadley et al, 2014). Copy number alterations
across tumor subtypes identified by pan-cancer analysis.
Some chromosomal changes, such as loss of 8p, occur across many tumor types. Other
changes occur in a select few tumor types. These changes include 3p loss and 3q gain in
squamous cell tumors such as lung squamous cell carcinoma (LUSC) and head and neck
squamous cell carcinoma (HNSC).
Figure 6. PCR identifies recombination of 3p arm.
Primers were designed in the endogenous 3p DNA sequence and telomere containing plasmid
– both outside the 1kb homologous region. For CRISPR #2, recombination was present in the
transfected cell population, as evidenced by PCR products on a gel (top right) and sequencing
of the PCR product (bottom).
Figure 7. Limiting dilution analysis for frequency of 3p recombination.
Cells were diluted to 100 or 1000 cells per well and presence of recombination was determined
by PCR. Frequency for cells with 3p recombination is ~1/300.
1650 bp
1000 bp
Figure 5. PCR identifies recombination of 8p arm.
Primers were designed in the endogenous 8p DNA sequence and telomere containing
plasmid – both outside the 1kb homologous region. For two CRISPRs (1 and 2),
recombination was present in the transfected 293T cell population, as evidenced by PCR
products on a gel (top right) and sequencing of the PCR product (bottom).
Untransfected
Puro Selected
1 1 2 2
Recombination
Plasmid Alone
PUROHR
HR PURO
1100 bp
PCR product
Recombined Chromosome 8
PUROHR
HR PURO
1300 bp
PCR product
Recombined Chromosome 3
1:300
1500 bp
1000 bp