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A CRISPR alternative for editing genes without cutting

Columbia University’s INTEGRATE system for gene editing could reduce the risk of off-target effects, because unlike CRISPR it doesn’t require DNA cuts. (Ernesto del Aguila III, National Human Genome Research Institute, NIH)

A well-known challenge facing scientists who want to use the CRISPR-Cas9 gene editing technology to address a range of diseases is that it requires cutting DNA, which can lead to errors. Scientists at Columbia University’s Vagelos College of Physicians and Surgeons are now proposing an alternative gene-editing system—one that sidesteps the need for DNA cutting altogether.

The researchers are using a “jumping gene,” or transposon, from a bacterium called Vibrio cholerae. The transposon is able to insert itself into different regions of the genome and can be programmed to carry any DNA sequence to any site. Therefore their technology, which they dubbed INTEGRATE, acts less like molecular scissors and more like molecular glue, they explained in the journal Nature.

“Rather than introduce DNA breaks and rely on the cell to repair the break, INTEGRATE directly inserts a user-defined DNA sequence at a precise location in the genome, a capability that molecular biologists have sought for decades,” said senior author Sam Sternberg, Ph.D., assistant professor of biochemistry and molecular biophysics at Columbia, in a statement. Sternberg recently joined Columbia after a stint working in the lab of CRISPR pioneer Jennifer Doudna at the University of California, Berkeley.

Because CRISPR is a process the occurs naturally in bacteria, Sternberg’s team decided to search for alternative gene editing systems in V. cholerae. They discovered that the jumping gene uses an enzyme to squeeze itself into the genome, rather than cutting the DNA. They then created a gene-editing system that combines that integration ability with a DNA payload and a guide RNA to help the tool find the right place to insert the edit.

From there, they sequenced the altered bacteria to confirm the edits were made correctly and that there were no copies of the inserted DNA payloads at unintended sites.

Despite the well-publicized concerns about CRISPR, enthusiasm for the technology hasn’t waned. In fact, just last week, Vertex Pharmaceuticals agreed to pay $420 million to acquire Exonics and to expand its partnership with CRISPR Therapeutics. The deal sets in motion a plan to use CRISPR to treat Duchenne muscular dystrophy and myotonic dystrophy type 1.

Nonetheless, academic labs around the world are pursuing multiple projects aimed at making gene editing safer. In May, a team at the Fred Hutchinson Cancer Research Center described a method developed there to use gold nanoparticles to carry CRISPR components into cells and to use the Cas12a enzyme to make cleaner cuts that Cas9 typically does. A UC Berkeley spinoff, GenEdit, is also developing a gold-based CRISPR system.

Other recently proposed ideas for improving CRISPR include attaching a hairpin-like guide to RNA to improve the accuracy of DNA cuts and adding an on-off switch to Cas9 enzymes to ensure they can’t make edits anywhere other than the targeted sites.

The next step for Sternberg’s team at Columbia is to test the INTEGRATE technology in mammalian cells. They believe the technique could eventually be applied to a variety of products, such as gene therapies and engineered crops.