Nucleic Acids Research Advance Access published online on October 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp757
Genome Integrity, Repair and Replication |
Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, 2Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095 and 3Neurology Service, VA Palo Alto Health Care Systems, Palo Alto, CA 94304, USA
*To whom correspondence should be addressed. Email: cbertoni{at}ucla.edu
Correspondence may also be addressed to Thomas A. Rando. Email: rando{at}stanford.edu
Received January 27, 2009. Revised August 21, 2009. Accepted August 28, 2009.
Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.