Researchers from Osaka University have advanced the field of gene editing by formulating a technique that significantly minimizes unintentional DNA mutations. Historically, the CRISPR/Cas9 method has been the standard in gene editing due to its ability to precisely correct mutations causing genetic diseases. However, this method is not without its pitfalls. The primary concern is the unintended mutations it can cause, potentially leading to adverse effects.
A recent publication in Nature Communications highlighted this new method, termed NICER. This approach leverages the capabilities of an enzyme known as a nickase, which produces multiple small cuts on single DNA strands.
The prevalent CRISPR/Cas9 method employs tiny segments of genetic code, termed guide RNAs, combined with the Cas9 enzyme. This pairing is tasked with identifying a specific DNA section where the Cas9 enzyme subsequently creates a disruption in the DNA's double-stranded structure. This disruption is essential for introducing modifications to the DNA. However, cells often repair these disruptions in ways that inadvertently introduce mutations. There's also a risk associated with the accidental addition of external DNA into the human genome, raising concerns about the safety of using CRISPR/Cas9 in clinical settings.
To address these issues, the researchers from Osaka University considered the use of Cas9 nickase. This enzyme produces single-strand disruptions in DNA, which are generally mended without triggering mutations.
Akiko Tomita, the study's lead author, explains, “Each chromosome in the genome has a ‘homologous’ copy. Using the NICER technique, heterozygous mutations—in which a mutation appears in one chromosome but not its homologous copy—are repaired using the unmutated homologous chromosome as a template.”
The researchers' preliminary tests were on human lymphoblast cells with an identified mutation in the TK1 gene. Subjecting these cells to nickase led to a minimal recovery in TK1 activity. By creating numerous interruptions in the TK1 area of both paired chromosomes, they amplified the gene correction efficiency nearly seventeen-fold through the activation of a cell repair process.
Senior author, Shinichiro Nakada adds, “Further genomic analysis showed that the NICER technique rarely induced off-target mutations. 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.”
The NICER technique's exclusion of DNA double-strand disruptions and the absence of external DNA suggest it may offer a safer alternative to traditional CRISPR/Cas9 methods. It holds promise as a new strategy for addressing genetic conditions resulting from heterozygous mutations.