Abstract
A study in four cell lines concludes that CRISPR/Cas9 editing causes unanticipated genomic alterations, including large deletions, translocations, and insertions. Some of these changes could be the first hits that cause cells to eventually become neoplastic.
CRISPR/Cas9 is already being used in clinical trials, but a recent study suggests that the genome-editing technology can cause large deletions and other unexpected genomic changes. Scientists say the results demonstrate the need to identify the genomic alterations produced by editing before applying the technology to patient care.
Scientists first reported that CRISPR/Cas9 could edit human genes in 2013. Since then, they have made several improvements to the technique to increase its specificity, leading regulatory agencies to give a nod to clinical trials that apply the method. The first trial began 2 years ago in China: Patients with advanced non–small cell lung cancer received their own T cells, which researchers had altered to remove the PD-1 receptor, thus preventing tumors from inhibiting T-cell action. The first U.S. trial, led by researchers at the University of Pennsylvania in Philadelphia, is recruiting patients with multiple myeloma and other cancers to receive edited T cells.
Allan Bradley, PhD, of the Wellcome Sanger Institute in the UK, and colleagues suspected that previous studies had missed genome damage from CRISPR/Cas9 because those studies checked only for changes near the editing site and for off-target effects. Bradley's group expanded the search and discovered that the method caused large changes around the target site in four different cell lines. When they employed CRISPR/Cas9 to delete PigA from mouse embryonic stem cells, for instance, sequencing the region around the gene showed frequent deletions spanning up to 6 kb. They also detected inversions, insertions, and other genomic damage.
To find out whether other cell types were similarly affected, the researchers used CRISPR/Cas9 to edit a different mouse embryonic stem cell line, a mouse bone marrow cell line, and a human retinal epithelium cell line. In all three, the scientists found that genomic alterations around the target site were prevalent. In the mouse bone marrow cell line, for example, more than one third of clones contained large deletions ranging from 100 bp to 3 kb.
The findings suggest that the changes likely result from cells' imprecise attempts to repair CRISPR/Cas9 cuts, says Michael Kosicki, the study's first author and a PhD candidate at the Wellcome Sanger Institute. The large genomic deletions and rearrangements the team discovered “could lead to proto-oncogenic changes,” he says. The genomic damage could activate cancer driver genes, constituting the initial “hits” that prompt cells to become neoplastic over time.
“It's an important study,” says Michael Boutros, PhD, of the German Cancer Research Center in Heidelberg, who wasn't connected to the research. “It shows that one should do a deep characterization of CRISPR modifications if one wants to use CRISPR/Cas9 in a clinical setting.”
The genomic damage the researchers detected “is a cause for concern” in clinical trials, says Thales Papagiannakopoulos, PhD, of the New York University School of Medicine in New York. He agrees that the careful analysis of mutations triggered by CRISPR/Cas9 cutting “has to become a basic step before putting anything back into patients.” –Mitch Leslie
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