New gene-editing technique allows
scientists to more rapidly study the role of mutations in tumor
development.
Anne Trafton | MIT News Office
Sequencing the genomes of tumor cells
has revealed thousands of mutations associated with cancer. One way
to discover the role of these mutations is to breed a strain of mice
that carry the genetic flaw — but breeding such mice is an
expensive, time-consuming process.
Now, MIT researchers have found an
alternative: They have shown that a gene-editing system called CRISPR
can introduce cancer-causing mutations into the livers of adult mice,
enabling scientists to screen these mutations much more quickly.
In a study appearing in the Aug. 6
issue of Nature, the researchers generated liver tumors in adult mice
by disrupting the tumor suppressor genes p53 and pten. They are now
working on ways to deliver the necessary CRISPR components to other
organs, allowing them to investigate mutations found in other types
of cancer.
“The sequencing of human tumors has
revealed hundreds of oncogenes and tumor suppressor genes in
different combinations. The flexibility of this technology, as
delivery gets better in the future, will give you a way to pretty
rapidly test those combinations,” says Institute Professor Phillip
Sharp, an author of the paper.
Tyler Jacks, director of MIT’s Koch
Institute for Integrative Cancer Research and the David H. Koch
Professor of Biology, is the paper’s senior author. The lead
authors are Koch Institute postdocs Wen Xue, Sidi Chen, and Hao Yin.
Gene disruption
CRISPR relies on cellular machinery
that bacteria use to defend themselves from viral infection.
Researchers have copied this bacterial system to create gene-editing
complexes that include a DNA-cutting enzyme called Cas9 bound to a
short RNA guide strand that is programmed to bind to a specific
genome sequence, telling Cas9 where to make its cut.
In some cases, the researchers simply
snip out part of a gene to disrupt its function; in others, they also
introduce a DNA template strand that encodes a new sequence to
replace the deleted DNA.
To investigate the potential usefulness
of CRISPR for creating mouse models of cancer, the researchers first
used it to knock out p53 and pten, which protect cells from becoming
cancerous by regulating cell growth. Previous studies have shown that
genetically engineered mice with mutations in both of those genes
will develop cancer within a few months.
Studies of such genetically engineered
mice have yielded many important discoveries, but the process, which
requires introducing mutations into embryonic stem cells, can take
more than a year and costs hundreds of thousands of dollars. “It’s
a very long process, and the more genes you’re working with, the
longer and more complicated it becomes,” Jacks says.
Using Cas enzymes targeted to cut
snippets of p53 and pten, the researchers were able to disrupt those
two genes in about 3 percent of liver cells, enough to produce liver
tumors within three months.
Many models possible
The researchers also used CRISPR to
create a mouse model with an oncogene called beta catenin, which
makes cells more likely to become cancerous if additional mutations
occur later on. To create this model, the researchers had to cut out
the normal version of the gene and replace it with an overactive
form, which was successful in about 0.5 percent of hepatocytes (the
cells that make up most of the liver).
The ability to not only delete genes,
but also to replace them with altered versions “really opens up all
sorts of new possibilities when you think about the kinds of genes
that you would want to mutate in the future,” Jacks says. “Both
loss of function and gain of function are possible.”
Using CRISPR to generate tumors should
allow scientists to more rapidly study how different genetic
mutations interact to produce cancers, as well as the effects of
potential drugs on tumors with a specific genetic profile.
“This is a game-changer for the
production of engineered strains of human cancer,” says Ronald
DePinho, director of the University of Texas MD Anderson Cancer
Center, who was not part of the research team. “CRISPR/Cas9 offers
the ability to totally ablate gene function in adult mice. Enhanced
potential of this powerful technology will be realized with improved
delivery methods, the testing of CRISPR/Cas9 efficiency in other
organs and tissues, and the use of CRISPR/Cas9 in tumor-prone
backgrounds.”
In this study, the researchers
delivered the genes necessary for CRISPR through injections into
veins in the tails of the mice. While this is an effective way to get
genetic material to the liver, it would not work for other organs of
interest. However, nanoparticles and other delivery methods now being
developed for DNA and RNA could prove more effective in targeting
other organs, Sharp says.
The research was funded by the National
Institutes of Health and the National Cancer Institute.
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