CRISPR RNA-guided nucleases (RGNs) have drummed up much excitement among scientists in the gene- and genome-editing fields. And for good reason: Studies have shown CRISPR-associated RGNs can precisely clip DNA strands, making room for new material to be added.

But researchers at Massachusetts General Hospital now advise additional caution—they show in a Nature Biotechnology report that CRISPR-associated RGNs readily produce off-target alterations.

“We found that expression of CRISPR-Cas RGNs in human cells can have off-target effects that, surprisingly, can occur at sites with significant sequence differences from the targeted DNA site,” co-author J. Keith Joung, M.D., Ph.D., associate chief for research at Mass General, said in a statement.

Using a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated RGNs, Dr. Joung and his colleagues identified off-target alterations induced by four of six RGNs aimed at endogenous loci by examination of partially mismatched sites. “The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site,” the researchers report.

Dr. Joung is quick to point out that these RGNs “continue to have tremendous advantages over other genome editing technologies,” he said, though notes that their precision must be improved before they are suitable for therapeutic applications.

“Our results don’t mean that RGNs cannot be important research tools, but they do mean that researchers need to account for these potentially confounding effects in their experiments. They also suggest that the existing RGN platform may not be ready for therapeutic applications,” Dr. Joung said. His team is now working to reduce off-target effects in an effort to improve future RGN approaches.

“I am optimistic that we can further engineer this system to achieve greater specificity so that it might be used for therapy of human diseases,” he added.

“High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells” appeared online in Nature Biotechnology June 23.

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