‘Harnessing evolution’

Gene mutations have consequences both good and bad — from resistance to conditions like diabetes to susceptibility to certain cancers.

In order to study these mutations, scientists need to introduce them directly into human cells. But changing genetic instructions inside cells is complex. The human genome comprises 3 billion base pairs of DNA divided across tens of thousands of genes.

To that end, Harvard researchers have created a tool that allows them to rapidly create mutations only in particular genes of interest without disturbing the rest of the genome. Described in Science, their tool, called Helicase-Assisted Continuous Editing (HACE), can be deployed to predetermined regions of the genome in intact, living cells.

“The development of tools like this marks a significant leap forward in our ability to harness evolution directly within human cells,” said first author Xi Dawn Chen, a Griffin Graduate School of Arts and Sciences student studying synthetic biology in the Department of Stem Cell and Regenerative Biology. “By allowing targeted mutagenesis in specific parts of the genome, this tool opens the door to creating enzymes and treatments that were previously out of reach.”

Unlike current methods for mutagenesis, which involve inserting extra copies of genes or broadly mutating many different genes at once, HACE offers the advantage of being directed to locations — like going to a specific address, rather than a neighborhood. The team’s novel bioengineering involves combining a helicase, which is an enzyme that naturally “unzips” DNA, with a gene-editing enzyme. They then use the gene-editing technology CRISPR-Cas9 to guide the protein pair to the gene they want to mutate. As the helicase unzips the DNA, it introduces mutations into only that gene sequence.

“HACE combines CRISPR’s precision with the ability to edit long stretches of DNA, making it a powerful tool for targeted evolution,” explained senior author Fei Chen, assistant professor in the Department of Stem Cell and Regenerative Biology and member at the Broad Institute.

To demonstrate the tool’s power in the lab, the scientists used it to identify drug resistance mutations in a gene called MEK1, which cancer treatments often target but frequently fail because the diseased cells mutate resistance mechanisms. Using HACE, the team sequenced only those mutated genes and pinpointed several unique changes associated with resistance to cancer drugs like trametinib and selumetinib, offering insights into how mutations affect drug performance.

They also examined how mutations in SF3B1, a gene involved in a biomolecular process called RNA splicing, affects RNA assembly. Mutations in this gene are common in blood cancers, but it’s been unclear which mutations cause the splicing defects; with HACE, the team could easily identify those changes.

And in partnership with Bradley Bernstein’s lab at Harvard Medical School and Dana-Farber Cancer Institute, the researchers also used the tool to better understand how changes in a regulatory DNA region affect the production of a protein in immune cells recognized as a potential target for cancer immunotherapies.

Bernstein said tools like HACE could someday allow massive edits of gene regulatory sequences that could then be coupled with deep learning computation for deciphering. “One can imagine many new therapeutic opportunities that involve precise edits or tuning of these regulatory sequences to ‘fix’ gene activity and ameliorate disease,” Bernstein said.

This research was supported by multiple sources including the National Institutes of Health, the Broad Institute, and the Harvard Stem Cell Institute.