Targeted gene knock in and sequence modulation mediated by a psoralen-linked triplex-forming oligonucleotide.
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.
Majumdar
Alokes
A
Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Muniandy
Parameswary A
PA
Liu
Jia
J
Liu
Ji-lan
JL
Liu
Su-ting
ST
Cuenoud
Bernard
B
Seidman
Michael M
MM
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005363:Ficusin / Q000494:pharmacology*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D004720:Endonucleases; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D005363:Ficusin