Researchers from Kyoto University say they have developed a alternative method to achieve scarless excision of selection markers and modify a single DNA base in the human genome with absolute precision. The technique, which is described in a Nature Communications paper published today, is unique in that it guides the cell's own repair mechanisms by design, providing pairs of genetically matched cells for studying disease-related mutations.
In order to understand the role of single nucleotide polymorphisms (SNPs) in hereditary disease, the team led by Knut Woltjen was looking at isogenic iPS cells, specifically isogenic "twins", cells whose genomes differ only by one SNP. According to Shin-Il Kim, an assistant professor in the Woltjen lab and co-first author on the study, creating these twins is not trivial. "Usually we need to add a gene for antibiotic resistance along with the SNP to overcome low efficiency. Since that adds another change to the genome, we also need a way to remove it."
To create isogenic twins, the Woltjen laboratory inserted a SNP modification along with a fluorescent reporter gene, which acts as a signal to detect modified cells. They also engineered a short duplicated DNA sequence, known as a microhomology, on the left and right sides of the reporter gene, and unique target sites for CRISPR.
These features allowed the researchers to exploit an endogenous DNA repair system in the cell called microhomology-mediated end joining (MMEJ) in order to precisely remove the reporter gene. MMEJ removes the fluorescent reporter gene, leaving only the modified SNP behind. By arranging the mutant SNP in one microhomology and the normal SNP in the other, the method efficiently generates isogenic twins.
The team named their new method MhAX, or Microhomology-Assisted eXcision. "To make MhAX work, we duplicate DNA sequences which are already present in the genome. We then let the cells resolve this duplication. At the same time, the cells decide which SNPs will remain after repair," Kim says. "One experiment results in the full spectrum of possible SNP genotypes."
To demonstrate the utility of their method, the Woltjen lab used MhAX to create SNPs in the HPRT and APRT genes, mutations that are associated with gout and kidney disease, respectively. Biochemical analyses showed cells with the HPRT mutant SNP had altered metabolism similar to patients, while the isogenic twin control cells, derived in the same experiment, were normal.