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Targeted Mutagenesis in the ‘Nickase’ of Time

Sarah C.P. Williams

A new kind of nickase has eliminated the concern for off-target mutations during genetic engineering.

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As the list of genetic diseases that could be cured by gene therapy continues to grow, a concern for scientists has been how to best deliver modified genes to patients. The challenges in this area are many—from delivery to preventing immune responses. But perhaps the most basic challenge is how to best replace an existing stretch of DNA with a new one—targeted mutagenesis. Existing methods of targeted mutagenesis, while effective, also have the side effect of changing other, related spots in the genome.

While standard nucleases lead to double strand breaks in DNA, nickases only cut one strand. This eliminates the side effect of off-target mutations. Source: Jin-Soo Kim

“There are usually dozens of potential off-target sites in the genome, because human genes are duplicated and repetitive by nature,” explained biologist Jin-Soo Kim of Seoul National University in South Korea. “If you accidentally induce mutations at those off-target sites, that can lead to cancer or disease so those off-target mutations are a very critical concern.”

Kim and his colleagues have put to rest that concern, however, with the development of a nickase, an enzyme that breaks a single strand of DNA at a time to introduce new genetic material. Because a nickase cuts only one strand—rather than standard nucleases that cut both at once—unwanted nicks will be quickly repaired by the cell’s built-in single strand break repair system. At the desired mutagenesis site, a new strand of DNA will be integrated into the genome by the repair system.

The South Korean research group developed the nickase by using the Fok1 nuclease domain that’s common to zinc finger nucleases, but modified it to induce only single-strand breaks. They tested the nickase’s activity by putting it into human embryonic kidney cells. If it successfully integrated its new DNA into the cell, a bioluminescent luciferase gene would be expressed.

While other research teams have recently developed their own nickases, Kim’s lab group is the first to study the effect of nickases on off-target mutations. “Our data clearly shows that off-target mutations are undetectable using this method,” says Kim.

The drawback of nickases, they found, was that they were slightly less effective at the target site than nucleases. While a double-strand break inducing nuclease worked 13% of the time, the nickase worked only around 3% of the time. “We think this is because in the cell, single strand break repair is naturally less efficient than homologous recombination,” says Kim.

The team plans to improve the efficiency by studying the role of key single strand break repair genes to the process. “Nucleases have been around for decades and been optimized,” he says. “Nickases have only been around a few months, so we still need time to improve their efficiency.”


  1. Kim E., Kim S., Kim D.H., Choi B-S., Choi I-Y., Kim J-S. 2012 Precision genome engineering with programmable DNA-nicking enzymes. Genome Research (April 20 online ahead of print)