Our method only uses oligonucleotides, short single-stranded DNA sequences of just 21 bases that are identical to the chromosomal region to be modified except for the central base designed to effectuate the desired base substitution. However, we have to deal with a strong cellular defense mechanism against accidental base-pair changes, the DNA mismatch repair system (abbreviated as MMR), which also effectively frustrates attempts to intentionally change a specific base-pair.
We found that MMR can be fooled when the central base in the oligonucleotide contains a simple chemical modification that does not affect its identity, but makes it invisible to MMR proteins. Our “oligo targeting” technique effectuates single base-pair substitution at a frequency of about one per 1000 cells, admittedly lower than reported efficacies of CRISPR/Cas9-assisted gene editing. But…oligo targeting has a strong advantage: extremely high precision.
This opens possibilities that are less feasible with current CRISPR/Cas protocols. One is “oligonucleotide-directed mutation screening” (ODMS), a rapid and high-throughput protocol we developed to interrogate so-called variants of uncertain clinical significance (VUS), which we are currently implementing in the clinical diagnostics of hereditary cancer.