Recent studies have identified a potential flaw in CRISPR, a form of genetic editing long heralded as a paradigm shift in its field. Nevertheless, Yale researchers still see a bright future for the technique.
CRISPR enables scientists to rewrite the genome with higher precision and more ease than ever before, but it is not without complications. This June, two separate studies published in the journal Nature Medicine found that cells edited with CRISPR are more likely to develop cancer — becoming ticking time bombs.
“[Complications from CRISPR can leave] the cell transistently vulnerable to the introduction of chromosomal rearrangement and other tumorigenic mutations,” one of the papers concluded. “This makes editing much more difficult.”
But Greg Licholai MED ’95, a lecturer at the School of Management and biotechnology entrepreneur, cautioned against making a blanket statement about the genome-editing technique.
“The tool needs to be well-understood. Any tool might have inadvertent consequences if it’s used in a way that’s not being fully understood,” Licholai said. “I think it’s inaccurate to make a blanket statement that CRISPR causes cancer — the same way it would be inaccurate to just say that cars kill people.”
CRISPR, short for clustered regularly interspaced short palindromic repeats, is a technique used to manipulate genetic material contained in cells. CRISPR systems use an enzyme called Cas9 to cleave DNA like a highly precise pair of molecular scissors. Then, researchers can take advantage of the cell’s repair mechanisms to knock out a sequence of interest or insert a new desired sequence in its place.
“Previously, when working with DNA, we could change chapters of the book of life,” Licholai said. “Now, with CRISPR, we can get down to literally changing the sequence of the letters and the words, which gives us a much greater kind of editing capability and precision.”
CRISPR experiments are much easier to set up than other gene-editing techniques, such as gene therapy and zinc finger nucleases, he added.
The two papers present one of the most formidable challenges to CRISPR-Cas9 genome editing since the technique’s discovery in 2012. In the teams’ experiments, after CRISPR-Cas9 cut both strands of the DNA double helix, a gene called p53 initiated a repair mechanism. This “policeman” caused the cell to either self-destruct — a process known as apoptosis — or fix the DNA break.
Because the cells die or fix the break, they do not successfully take up the CRISPR edits. Thus, the p53 mechanism makes CRISPR highly inefficient — reducing efficiency seventeenfold, according to one of the teams.
More dangerously, the cells that are successfully altered do so because their p53 protein is dysfunctional. That protein suppresses tumors, and its dysfunction can cause cancer.
“That CRISPR-edited cells downregulate an important tumor suppressor gene, p53, is important to be known by the field,” said Sidi Chen, a genetics professor at the medical school.
Mutations in p53 cause about half of ovarian cancers, over a third of lung cancers and almost a third of pancreatic, stomach and liver cancers, according to a 2010 paper in the journal Cold Spring Harbor Perspectives in Biology.
The studies published in June concluded that in creating cell-based CRISPR-Cas9 therapies, scientists must ensure that genome-edited cells still have a functional p53 gene.
There are still several ways to edit successfully with CRISPR, Chen noted. These include using high-fidelity enzymes to reduce off-target effects — unintended genetic modifications — or newer techniques such as CRISPR interference to enhance efficiency and specificity.
Additionally, the media has misconstrued the findings of these papers, said Antonio Giraldez, the chair of genetics at the School of Medicine, who owns stocks in CRISPR Therapeutics and Editas Medicine, two companies that focus on CRISPR-based therapies.
“The media has modified the punchline of the studies by saying that CRISPR causes cancer,” Giraldez said. “That’s a complete misconception on the reading of the papers.”
The findings do not present a major hurdle to CRISPR, he added.
For example, researchers can also inhibit p53 in ways that aren’t permanent in order to prevent cell death. Scientists must just be cautious that cells selected with the mutations on the gene of interest by CRISPR are not also mutants for the p53 gene, he said.
“If the experiments are not performed carefully, what you could be selecting for are cells that survive upon CRISPR activity — and those cells might be mutants of the p53 tumor suppressor gene,” he said. “So, I think these studies are quite enlightening in light of the hurdles that have to be overcome in the road toward genetic modification using CRISPR.”
Giraldez’s lab, which studies CRISPR using zebrafish as a model organism, has not seen evidence of p53 selection, he said. He suggested that the lab’s ability to modulate the level of Cas9 used may prevent this phenomenon and that embryonic stem cells — used in one of the two studies — might have enhanced activity of p53.
Serious risks still remain for CRISPR. DNA damage and off-target effects are some of the most important concerns, Licholai said. And because CRISPR has developed so quickly, scientists still don’t fully know its reliability or side effects, he said.
Despite the hurdles that have to be overcome, the researchers are optimistic about the progression of CRISPR research. The road for CRISPR to enter the clinic is likely shorter and smoother than previous gene-editing therapies, Giraldez said.
“The potential for this technology is outstanding, as long as the specificity is high and scientists take care to make sure there are no additional mutations introduced to the cells,” he said. “And I think that is possible.”
Amy Xiong | firstname.lastname@example.org