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

Abstract

Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo. Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, 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 coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively. The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…Early models for ssODN-mediated gene targeting proposed that the ssODN annealed to its chromosomal complement and served as a template for repair of this chromosomal DNA strand ( Figure 1, Model I; see also Andersen et al 42 and Liu et al 43 for review). As chimeric RNA/DNA oligonucleotides exhibited reduced gene repair frequencies in human cell-free extracts lacking hMSH2 protein, 44 it was postulated that the MMR proteins were responsible for copying MutSa mainly recognizes single base-base mismatches and small loops of one or two nucleotides, whereas MutSb has more affinity for larger loops of two-five unpaired nucleotides. Upon mismatch binding and recruitment of the MLH1/PMS2 heterodimer (MutLa), the complex undergoes an ATPdriven conformational switch, which allows sliding away from the mismatch.…”

Commentary: Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

“…It is referenced in the literature under other names such as targeted nucleotide exchange, chimeraplasty, oligonucleotidemediated gene editing, chimeric oligonucleotidedependent mismatch repair, oligonucleotide-mediated gene repair, triplex-forming oligonucleotides induced recombination, oligodeoxynucleotide-directed gene modification, therapeutic nucleic acid repair approach, targeted gene repair (see e.g. Andersen et al, 2002;Christensen et al, 2006;Cole-Strauss et al, 1999;de Semir and Aran, 2006;Igoucheva et al, 2006;Zhang et al, 1998).…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…However, reported rates of gene repair have been highly variable (ranging from zero to ca. 40%), and occasional unexpected mutational effects have been found [11,50]. These problems have engendered a certain amount of controversy concerning the use of COs in gene targeting, particularly in the context of gene therapy.…”