MIT Team Uses Prime Editing to Create Mouse Models of Cancer Mutations

MIT researchers have developed a way to more easily engineer specific cancer-linked mutations into mouse models. Using prime editing, the team created models of several different mutations of the cancer-causing gene Kras in different organs. They believe this technique could also be used for nearly any other type of cancer mutation that has been identified.

“This is a remarkably powerful tool for examining the effects of essentially any mutation of interest in an intact animal, and in a fraction of the time required for earlier methods,” says Tyler Jacks, one of the senior authors of the study published in Nature Biotechnology.

Researchers have traditionally used genetic engineering to create mouse models by deleting tumor suppressor genes or activating cancer-promoting genes. However, this approach is labor-intensive and requires several months or even years to produce and analyze mice with a single cancer-linked mutation.

More recently, researchers began exploring the possibility of using the CRISPR genome-editing system to make cancerous mutations more easily. However, while this approach makes it easy to knock out genes, it doesn’t lend itself to inserting new mutations into a gene because it relies on the cell’s DNA repair mechanisms, which tend to introduce errors.

In the current study, the team turned to prime editing, developed in the Liu lab at the Broad Institute, as a way to perform more precise gene-editing that would allow them to make very targeted mutations to either or tumor suppressors.

They designed new mouse models by engineering the gene for the prime editor enzyme into the germline cells of the mice, which means that it will be present in every cell of the organism. The encoded prime editor enzyme allows cells to copy an RNA sequence into DNA that is incorporated into the genome. However, the prime editor gene remains silent until activated by the delivery of a specific protein called Cre recombinase.

Since the prime editing system is installed in the mouse genome, researchers can initiate tumor growth by injecting Cre recombinase into the tissue where they want a cancer mutation to be expressed, along with a guide RNA that directs Cas9 nickase to make a specific edit in the cells’ genome. The RNA guide can be designed to induce single DNA base substitutions, deletions, or additions in a specified gene, allowing the researchers to create any cancer mutation they wish.

To demonstrate the potential of this technique, the researchers engineered several different mutations into the Kras gene, which drives about 30 percent of all human cancers. However, not all Kras mutations are identical. Many Kras mutations occur at a location known as G12, where the amino acid glycine is found, and depending on the mutation, this glycine can be converted into one of several different amino acids.

The researchers developed models of four different types of Kras mutations found in lung cancer: G12C, G12D, G12R, and G12A. To their surprise, they found that the tumors generated in each of these models had very different traits. For example, G12R mutations produced large, aggressive lung tumors, while G12A tumors were smaller and progressed more slowly.

The researchers also used their technique to create pancreatic organoids with several different types of mutations in the tumor suppressor gene p53, and they are now developing mouse models of these mutations. They are also working on generating models of additional Kras mutations, along with other mutations that help to confer resistance to Kras inhibitors.

The team has made mice with the prime editing system engineered into their genome available through a repository at the Jackson Laboratory, and they hope that other labs will begin to use this technique for their own studies of cancer mutations.