A journey back in time helps solve a modern gene editing conundrum

April 18, 2024

By Christopher Vaughan

Hiro Nakauchi, MD, PhD

The gene editing technology known as CRISPR-Cas9 has given researchers the opportunity to precisely edit genes, an ability that can potentially lead to prevention or cures for genetic diseases that have long been incurable. However, researchers at the Institute for Stem Cell Biology and Regenerative Medicine found—somewhat by accident—that the gene editing technology is often not as precise as scientists think, and that some of the errors that it produces are difficult to spot using current technologies. Straightening things out required institute scientists to use older genetic technology common a generation ago. 

The problem was first noticed by Fabian Suchy, PhD, a postdoctoral fellow in the laboratory of Professor Hiro Nakauchi, MD, PhD. After using the CRISPR system with engineered viral particles to carefully insert new genes into cells, he noticed that the cells’ appearance and behavior (the phenotype) was inconsistent with indications of whether the right genes had been successfully inserted (the genotype). Cells that showed fluorescent labels indicating that they had successfully taken up the inserted gene were not acting like it.

“We weren’t sure what was going on,” Suchy said. “At first we thought that the genes were sometimes being randomly inserted (and were therefore not active), but that turned out not to be the case.”

Sequencing some gene-edited cells’ DNA showed that the new genes were indeed inserted where they should be. Suchy did some digging into the historical record and found something interesting. Normally, the new genes the researchers intend to edit into a cell’s genome are nested in the middle of leftover viral DNA sequences that are intended to drop away as the gene is inserted into its target position. But through a biochemical quirk, the bits of viral DNA that were used to carry the new gene into the genome could link up with each other to form long chains. If the bits of viral DNA link up with each other, it could create an insertion that includes multiple copies of the new gene interspersed with bits of viral DNA between each one. 

“The linkage of this viral DNA was shown in the 1970s, but these genetic chains have not been shown to frequently insert by researchers doing gene editing now,” Suchy said.

Proving that this linkage was happening in the lab—and that the scientists were inserting multiple genes and viral material instead of single genes—was not simple, however. “It was very hard to spot the insertion of multiple genes using common, modern methods,” Suchy said.

To find out whether this was happening, Suchy enlisted the help of senior research scientist Katja Pekrun, PhD. Pekrun is familiar with Southern blots, a technique invented in the 1970s that was well known to any genetic researcher practicing 40 years ago, but which is now largely unused. Southern blots break up DNA into pieces and then use an electric current to pull those pieces through a gel. Large pieces of DNA move more slowly than small pieces, so if multiple copies of the gene and viral material are inserted, that section of DNA will not advance as far in the gel over time.

“The Southern blots were tedious to do, and required a lot of DNA,” Suchy said. “But once we finished, it was very obvious that multiple genes were being inserted.” They eventually developed easier detection methods and found that around 50 percent of the edited cells had these hidden, repeat insertions.

Once this viral DNA linkage and insertion was identified in one system, Suchy teamed up with postdoctoral fellow Daiki Karigane MD, PhD in the lab of institute director Ravi Majeti MD, PhD to investigate this phenomenon in many cell types and genes. Indeed, the team found these linked insertions could occur in multiple genes and cell types, including pluripotent stem cells and blood stem cells, indicating this to be a broad problem in gene editing, the researchers said.

Luckily, the team also went on to find ways to avoid the viral DNA linking that lead to multiple gene insertions. A key element of editing with CRISPR is the use of a guide RNA that directs a protein called Cas9 to cut the cell’s DNA at a precise location. The team found that they could use a different guide RNA to cut out the viral DNA before the new gene was inserted. This reduced the incidence of multiple insertions by more than ten-fold, they said. 

“These findings might help researchers around the world who are making gene edits understand why they may not be getting the results they expect, and give them tools to correct the problem,” Nakauchi said. 

The research was published in the Journal Nature Biotechnology. Suchy and Karigane were co-first authors on the paper. Nakauchi and Majeti were co-senior authors on the paper.