CRISPR Used to Create Mouse Models

Thousands of cancer-associated mutations have been discovered through tumor genome sequencing. A common strategy to study a particular mutation requires scientists to create and breed a strain of mice that carries the aberrant gene, a time-consuming and costly process.

A faster, less expensive method may now be possible, according to new research from the Massachusetts Institute of Technology (MIT) in Cambridge. Using a genome-editing system called CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) that protects bacteria from phage infections, scientists altered the tumor suppressor genes Pten and p53 in about 3% of liver cells in mice. This was enough to produce tumors within 3 months, researchers reported (Nature 2014 Aug 6 [Epub ahead of print]).

“The beauty of this system is speed,” says senior author Tyler Jacks, PhD, director of MIT's Koch Institute for Integrative Cancer Research. “The alternative process might take a year or more to get the same answer that we could get with this system in weeks.”

This new method of cancer-model generation includes an enzyme called Cas9 that binds to and cuts DNA, and a short RNA guide strand that leads Cas9 to the DNA target.

MIT researchers used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single-guide RNAs to the liver that directly targeted Pten and p53. Cas9 snipped the DNA precisely where researchers engineered the break to occur. “When the break is improperly repaired, a mutation results, which is what we were aiming for,” says Jacks.

Targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-LoxP technology–mediated deletion of the two genes. In addition, researchers also used the CRISPR/Cas system to cut out the normal version of the β-catenin oncogene and replace it with a form containing activating mutations. The genetic switch was successful in about 0.5% of hepatocytes.

Being able to both replace a gene and mimic its deletion is important because cancer gene mutations fall into both categories, Jacks says. “Some are loss of function, some are gain of function. This editing ability is critical for accurately modeling certain types of cancer-associated mutations.”

Injecting CRISPR components into veins in the tails of mice is an effective method for getting genetic material to the liver, a natural destination for foreign material filtered from the blood. Jacks's lab is now working on methods to deliver CRISPR components to other organs. From the long list of potential cancer genes, scientists will be able to rapidly evaluate the role each plays in tumor development and identify driver mutations.

“The faster we know the drivers, the faster we'll be able to develop and test new medicines that are directed at the genes or the proteins produced by those mutations,” says Jacks.

In the future, he says, this genome-editing tool may even make it possible to correct cancer-causing mutations. “If you can do genome editing in vivo, which is what we've done, could you correct cancer-predisposing mutations in humans?” Jacks wonders. “That's not going to happen tomorrow, but down the road, it's not out of the realm of possibility.”

For more news on cancer research, visit Cancer Discovery online at http://CDnews.aacrjournals.org.