When scientists study the genetics of cancer, they often breed mice strains that carry selected cancer-associated mutations. But cultivating such strains, usually via transgenesis or gene targeting in embryonic stem cells, is often time-consuming and expensive. Could there be a better way—a faster, cheaper way—to create mice strains that carry particular genetic flaws?
An alternative has been proposed by researchers from MIT. They have shown that the CRISPR gene editing system can introduce cancer-causing mutations into the livers of adult mice. The researchers anticipate that their method will allow for more rapid testing of any single genes or gene combinations that are suspected of being capable of initiating tumor formation in the liver.
“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,” said Phillip Sharp, Ph.D., a professor at MIT’s Koch Institute for Integrative Cancer Research.
Dr. Sharp was part of the MIT research team, which was led by Koch Institute director Tyler Jacks, Ph.D. Dr. Jacks noted that the CRISPR technique, which not only provides the ability to 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.” Both loss of function and gain of function, he explained, are possible.
The MIT researchers presented their results August 6 in Nature, in an article entitled, “CRISPR-mediated direct mutation of cancer genes in the mouse liver.” It described how cancer models were generated using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice.
“We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumor suppressor genes Pten and p53 (also known as TP53 and Trp53), alone and in combination,” wrote the authors. “CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53.”
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. Using Cas enzymes targeted to cut snippets of p53 and Pten, the researchers were able to disrupt those two genes in about 3% of liver cells, enough to produce liver tumors within three months.
With traditional techniques, genetically engineering such models is “a very long process,” commented Dr. Jacks. “And the more genes you’re working with, the longer and more complicated it becomes.
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% of hepatocytes.
In the Nature article, the authors emphasized that simplified methods of testing the oncogenic properties of candidates in vivo are critical. In particular, they cited the need to somehow evaluate the thousands of candidate cancer genes that are being discovered through next-generation sequencing efforts.
Already looking forward to refining their method of generating cancer models, the authors suggested that it could attain greater sensitivity if CRISPR/Cas9-mediated mutagenesis could be performed on a “sensitized” background carrying constitutive or conditional mutations in a tumor suppressor gene such as p53. “More efficient delivery techniques, such as adenovirus or adeno-associated virus, more potent sgRNAs, and longer homologous recombination templates,” they wrote, “might also improve the overall efficiency of this method and expand the range of tissue that could be targeted.”