In 1993 and 1994, my laboratory was investigating the role of RNA in extending the half-life of DNA molecules conjoined at homologous regions during the initial phase of recombination known as synapsis. 1, 2 We observed that the length of complementary sequence required for stable hybridization during DNA pairing was reduced when one of the partners contained RNA. Since we believed that DNA pairing was the rate-limiting step of homologous recombination, we reasoned that controlling this phase might improve the frequency of gene targeting in cells from various origins.

Previous work in mammalian cultured cells had demonstrated that gene targeting or gene replacement was a rare event (see Ref. 3 for review) and significant barriers to success had been established by other workers in the field. This information prompted us to formulate the alternative strategy, known as targeted gene repair. In essence, we envisioned repairing a specific mutation rather than replacing an entire dysfunctional gene. Similar strategies using site-directed nucleotide conversion had been attempted in yeast, 4 but were believed to be target-and context-dependent. In our case, however, a novel chimeric oligonucleotide (Figure 1) containing regions of RNA to enhance binding affinity was designed as a tool to facilitate this process. Conceptually, the oligonucleotide would localize to the target site and form a stable complex. A DNA region containing a single ‘incorrect nucleotide’would be placed between the two RNA bridges, producing the mismatched base pair and a distorted helical structure upon binding. The mispaired bases would be recognized by a genetic repair system, which converts the targeted base in the genome using information provided by the chimeric oligonucleotide template.