Off-target mutations resulting from conventional genome editing methods. Due to the mutagenic nature of DNA double-strand break repair, even if the gene mutation is successfully corrected, there is a heightened risk of off-target mutations in areas distinct from the original target of CRISPR/Cas9. Credit: S. Nakada
Key points:
- Researchers in Japan have developed an alternative to CRISPR-Cas9 that significantly reduces unintended mutations.
- The new method relies on Cas9 nickase, which creates single-strand breaks rather than double.
- A recent study showed the method enhanced gene correction efficiency approximately 17-fold.
While CRISPR-Cas9 has been a boon for many areas of research, allowing scientists to make precise and impactful changes to an organism’s DNA, the method can also result in unintended DNA mutations that may have negative effects.
In Japan, researchers have developed a new gene editing technique that is as effective as CRISPR-Cas9, while significantly reducing the unintended mutations.
The novel technique, called NICER, is based on the creation of multiple small cuts in single DNA strands by an enzyme, called a nickase.
Traditional CRISPR-Cas9 editing uses guide RNAs to target a specific section of DNA, and the Cas9 enzyme initiates a break in the double-stranded DNA structure at this location. This double-strand break is key for initiating changes to the DNA. However, cellular repair of double-strand breaks can lead to unintended DNA mutations, as well as the integration of exogenous DNA to the human genome, which raises safety concerns.
To minimize these unintended mutations, the Osaka University-led research team investigated the use of Cas9 nickase, which creates single-strand breaks or “nicks” in DNA that are typically repaired without causing mutations.
For their initial experiments, the research team used human lymphoblast cells with a known heterozygous mutation in a gene called TK1. When these cells were treated with nickase to induce a single cut in the TK1 region, TK1 activity was recovered at a low rate. However, when the nickase induced multiple nicks in this region on both homologous chromosomes, gene correction efficiency was enhanced approximately 17-fold via activation of a cellular repair mechanism.
“Further genomic analysis showed that the NICER technique rarely induced off-target mutations,” said senior author Shinichiro Nakada. “We were also pleased to find that NICER was able to restore the expression of disease-causing genes in cells derived from genetic diseases involving compound heterozygous mutations.”