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Powerful gene-editing tool appears to cause off-target mutations in human cells
Date:6/23/2013

ully used CRISPR-Cas RGNs to make genomic changes in fruit flies, zebrafish, mice and in human cells including induced pluripotent stem cells which have many of the characteristics of embryonic stem cells. The technology's reliance on such a short RNA segment makes CRISPR-Cas RGNs much easier to use than other gene-editing tools called zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and RGNs can be programmed to introduce several genetic changes at the same time.

However, the possibility that CRISPR-Cas RGNs might cause additional, unwanted genetic changes has been largely unexplored, so Joung's team set out to investigate the occurrence of "off-target" mutations in human cells expressing CRISPR-Cas RGNs. Since the interaction between the guiding RNA segment and the target DNA relies on only 20 nucleotides, they hypothesized that the RNA might also recognize DNA segments that differed from the target by a few nucleotides.

Although previous studies had found that a single-nucleotide mismatch could prevent the action of some CRISPR-Cas RGNs, the MGH team's experiments in human cell lines found multiple instances in which mismatches of as many as five nucleotides did not prevent cleavage of an off-target DNA segment. They also found that the rates of mutation at off-target sites could be as high or even higher than at the targeted site, something that has not been observed with off-target mutations associated with ZFNs or TALENs.

"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," says Joung, who is an associate professor of Pathology at Harvard Medical School. "We are now working on ways to reduce these off-target effects, along with methods to identify all potent
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Contact: Sue McGreevey
smcgreevey@partners.org
617-724-2764
Massachusetts General Hospital
Source:Eurekalert

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