Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in amammalian cell-free extract

Chimeric oligonucleotides consisting of RNA and DNA residues have been show n to catalyze site-directed genetic alteration in mammalian cells both in v itro and in vivo. Since the frequency of these events appears to be logs hi gher than the rates of gene targeting, a process involving hamologous recom bination, we developed a system to study the mechanisms of chimera-directed gene conversion. Using a mammalian cell-free extract and a genetic readout in Escherichia coil, we find that point mutations and single base deletion s can be corrected at frequencies of similar to 0.1% and 0.005%, respective ly. The reaction depends an an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studie s reported herein suggest that the process of mismatch repair functions in site-directed gene correction.