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J. Biol. Chem., Vol. 283, Issue 17, 11244-11252, April 25, 2008

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Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide^*

Alokes Majumdar, Parameswary A. Muniandy, Jia Liu, Ji-lan Liu, Su-ting Liu, Bernard Cuenoud, and Michael M. Seidman¹

From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224 and Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland

Information from exogenous donor DNA can be introduced into the genome via homology-directed repair (HDR) pathways. These pathways are stimulated by double strand breaks and by DNA damage such as interstrand cross-links. We have employed triple helix-forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA interstrand cross-link at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock in of the donor at frequencies 2–3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1-XPF, which is involved in recombinational pathways as well as cross-link repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10-fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1-XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock in. We asked whether there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of double strand breaks into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways.

Received for publication, January 23, 2008 , and in revised form, February 21, 2008.

^* This work was supported by the Intramural Research Program of the NIA, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and supplemental references.

¹ To whom correspondence should be addressed: LMG/NIA/National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8565; Fax: 410-558-8157; E-mail: seidmanm{at}grc.nia.nih.gov.

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