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Researchers from the ӳ����ý and Columbia University have developed a way to insert entire healthy genes into human cells efficiently enough for potential therapeutic applications. It lays a foundation for gene-editing therapies for patients with different mutations that cause a genetic disease.

The approach uses laboratory-evolved versions of enzymes called CRISPR-associated transposases, or CASTs, which move large stretches of DNA in bacterial genomes but have so far shown minimal activity in mammalian cells.

The evolved CAST variants are capable of inserting thousands of DNA letters, or base pairs, into a target site in the genome of human cells, and are hundreds of times more efficient than natural CAST systems reported to date. The evolved CASTs could one day allow researchers to create gene editing therapies that precisely insert an entire healthy gene copy and hence benefit multiple patients with a genetic disease, regardless of their specific disease-causing mutation in that gene.

The work appears today in and comes from the labs of co-senior authors David Liu, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the ӳ����ý as well as a professor at Harvard University, and , who is an associate professor at Columbia. Both Sternberg and Liu are Howard Hughes Medical Institute investigators. 

Isaac Witte and Simon Eitzinger, both graduate students in Liu’s lab, and George Lampe, a postdoctoral scientist in Sternberg’s lab, are co-first authors on the work.

“This was a swing-for-the-fences project in that we started with a really complicated system that had a hundred-fold lower efficiency than would be therapeutically useful,” said Liu. “I’m really excited that we got there and I can’t wait to see what’s next.”

Protein evolution

The ability to install entire genes in cells precisely and efficiently has been a long-standing challenge in genome editing. In 2019, researchers including Sternberg and ӳ����ý’s Feng Zhang independently CASTs, which are found in bacteria, offering one potential step toward this goal. CASTs are enzymes that are guided by RNA and move large sections of DNA to new locations in a highly specific manner. They also do not create free double-strand breaks in the DNA, which can lead to unwanted editing byproducts.

But when scientists in Sternberg’s lab applied these enzymes in human cells, they found that editing occurred in only about 0.1 percent of cells, an efficiency too low to be useful as a potential therapeutic. 

To address this, Liu and Sternberg teamed up to harness the power of , a protein evolution approach developed by Liu’s lab in 2011. Using PACE, the researchers evolved  a CAST system from Pseudoalteromonas bacteria. After performing hundreds of rounds of evolution, they generated a system called evoCAST that was hundreds of times more efficient than the original CAST system in mammalian cells.

“In this work, we discovered that a key bottleneck for CAST in human cells was its limited transposition activity,” said Witte. “Now that we’ve addressed this bottleneck through laboratory evolution, CAST can excel as a genome editing tool in human cells, in large part due to its origins as a naturally existing system for RNA-guided gene integration.”

In human cells, evoCAST successfully installed genes relevant for diseases such as Fanconi anemia and phenylketonuria, and for improved CAR-T cell immunotherapy at efficiencies between 10 and 20 percent. The team is currently working to further develop evoCAST for use in therapeutically relevant settings and to apply their newly established CAST PACE platform to other CAST systems found in nature. 

Another gene-editing technique called eePASSIGE, developed by the Liu lab last year, also enables targeted gene integration, but Liu says evoCAST offers complementary strengths because it relies on a distinct mechanism of gene integration. eePASSIGE is generally more efficient, but evoCAST edits with high purity. evoCAST also installs genes in a single step, which could help scientists apply it more quickly than eePASSIGE, which integrates new DNA in two steps and requires a prime editor and a recombinase enzyme. 

“Mobile genetic elements like CAST offer a glimpse into nature’s ingenuity in programmable genome manipulation,” said Sternberg. “With evoCAST, we’ve shown how laboratory evolution can transform these naturally occurring systems into powerful tools for therapeutic gene insertion — and we believe we’ve only scratched the surface of what will be possible in the future.”