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. 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. Double strand breaks (DSBs) 2The abbreviations used are: DSB, double strand break; HDR, homology-directed repair; pso-TFO, psoralen-linked triple helix-forming oligonucleotide; SSO, single strand oligonucleotide; SSA, single strand annealing; Hprt, hypoxanthine phosphoribosyl transferase; 6-TG, 6 thioguanine; UVA, long wave ultraviolet light; CHO, Chinese hamster ovary. 2The abbreviations used are: DSB, double strand break; HDR, homology-directed repair; pso-TFO, psoralen-linked triple helix-forming oligonucleotide; SSO, single strand oligonucleotide; SSA, single strand annealing; Hprt, hypoxanthine phosphoribosyl transferase; 6-TG, 6 thioguanine; UVA, long wave ultraviolet light; CHO, Chinese hamster ovary. are among the most dangerous forms of DNA damage and may result in deletion, rearrangement of chromosomal sequences, or, if unrepaired, chromosome loss and possibly cell death (1van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). There are multiple pathways for DSB repair that are distinguished by the identity of the proteins and enzymes involved and their potential for mutagenesis of the break site (2Stark J.M. Pierce A.J. Oh J. Pastink A. Jasin M. Mol. Cell. Biol. 2004; 24: 9305-9316Crossref PubMed Scopus (357) Google Scholar, 3Lin Y. Lukacsovich T. Waldman A.S. Mol. Cell. Biol. 1999; 19: 8353-8360Crossref PubMed Scopus (72) Google Scholar, 4Sonoda E. Hochegger H. Saberi A. Taniguchi Y. Takeda S. DNA Repair (Amst.). 2006; 5: 1021-1029Crossref PubMed Scopus (375) Google Scholar, 5Couedel C. Mills K.D. Barchi M. Shen L. Olshen A. Johnson R.D. Nussenzweig A. Essers J. Kanaar R. Li G.C. Alt F.W. Jasin M. Genes Dev. 2004; 18: 1293-1304Crossref PubMed Scopus (123) Google Scholar, 6Weinstock D.M. Nakanishi K. Helgadottir H.R. Jasin M. Methods Enzymol. 2006; 409: 524-540Crossref PubMed Scopus (119) Google Scholar, 7Dronkert M.L. Beverloo H.B. 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Homologous recombination repair involves invasion of a DNA duplex by a single strand end and can be error-free or mutagenic depending on the sequence of the invaded duplex. Because the sequences with which they interact need not be absolutely identical to the single strand end, the homology-directed pathways provide an opportunity to manipulate the sequence of the genome. Gene conversion and recombination with exogenous double and single strand donor DNAs occur at impractically low frequencies in mammalian cells (9Vasquez K.M. Marburger K. Intody Z. Wilson J.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8403-8410Crossref PubMed Scopus (253) Google Scholar). However, DSBs in the genome markedly enhance the frequency of cis and trans recombination/gene conversion, as demonstrated in experiments with the homing endonuclease I-SceI (3Lin Y. Lukacsovich T. Waldman A.S. Mol. Cell. Biol. 1999; 19: 8353-8360Crossref PubMed Scopus (72) Google Scholar, 10Liang F. Han M. Romanienko P.J. Jasin M. 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In one used a double strand in the other employed single strand oligonucleotide (SSO) We cell with the sequence with both donors and have the of repair on the of the two Chinese hamster cell the cell and and B. F. A.J. 1997; PubMed Scopus Google cells were in with and to cells were in to cells. were in by a of the by A. B. F. A. A.J. J. Biol. Chem. 2003; 278: Full Full PubMed Scopus Google Scholar, M. K. H. K. Res. 1990; PubMed Scopus Google Scholar). cells were at and the the to with and the cells were and with for to in the cells were from the by with and the hamster was and as A. B. F. A. PubMed Scopus Google Scholar). were introduced by an In experiments with the donor nucleic of the duplex donor of an SSO donor were with the pso-TFO. was by for and in the to for at The cells were a and in were also in to were and the frequencies as the of cells. In experiments with double was to in to Gene In and at an I-SceI cells in were with an I-SceI and an SSO the duplex donor. were in for and in as of in were and in DNA was and the target region and which a with DNA from cells. of the with the was used to the frequency of sequence conversion by or other SSO. in of the duplex donor was by with a of and and a of and DNA from was on to and to a complementary to the region The frequencies of are on the of cells in the and The a to that can the of a A. A. J. J. Z. Y. J. Nat. Genet. 1998; 20: PubMed Scopus Google Scholar). sequence in a that is a sequence for interstrand cross-link by psoralen The pso-TFO, used in these experiments a of B. F. D. F. R.M. Chem. 1998; PubMed Scopus Google with the as A. B. F. A. PubMed Scopus Google Scholar, A. B. 2004; PubMed Scopus Google Scholar). of cells in resulted in the of A. B. F. A. A.J. J. Biol. 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However, be that the for a would the of sequences at the target site. asked whether the same be used to the frequency of mutagenesis of the target site in cells. We with the pso-TFO, a SSO to the target site the site with an In a of experiments the frequency of with the was However, when the donor was with the pso-TFO, the frequency of was greater of the DNA from the of the site by in of the such that the sequence conversion frequency was of the sequence of the target region in that the sequence from the the other an single from the donor sequence the cross-link target site These results a of donor sequence the of on the sequence. We the by of the and an SSO the sequence into the cells. resulted in a in to The of result was that DSB that would have deletions were by the SSO to sequence. for sequence were on the or loss of of the to the of of cells, DNA was the target sequence region and the by There are of due to donor oligonucleotides or of DNA Maher Dev. 1999; PubMed Scopus Google Scholar, D. J.M. 2003; PubMed Scopus Google Scholar, R. Y. D. J. C. D.C. 2006; PubMed Scopus Google Scholar). that the of donor oligonucleotide would have been to the between and experiments to The SSO was introduced into cells in the of the pso-TFO. The cells were for a and and as to of were DNA the target region and the by were In a DNA was from by of cells with the donor of the DNA was by target region and was with to single-stranded to and between the two were in the frequency of long of and the of the that the conversion of the target site was of DNA Repair on SSO and were into cells deficient in and ERCC1-XPF, by The sequence conversion results with the cells were to those with the in that the frequency of sequence was to cells However, in contrast to the results with the in cells the frequency of sequence by to sequence conversion by the SSO not We also the of the in cells deficient in and the repair proteins and in the experiments with the sequence conversion frequencies were unaffected by these between and Double DNA for from in HDR of a DSB is that single strand in a These a as would occur knock in of sequence from the long duplex donor as by the F. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, F. 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We that the frequency of sequence was the same as in the experiments with the cells were with the I-SceI and the donor or both the and SSO The knock-in frequency in cells with the I-SceI and the donor was greater than in the experiments with the both donors were the of donor was unaffected by the of the as in the with the the frequency of knock in of the duplex donor was not by the of the SSO donor. In deletion mutagenesis at the site of the target and that these were the consequence of DSBs formed the to the cross-link A. B. F. A. A.J. J. Biol. Chem. 2003; 278: Full Full PubMed Scopus Google Scholar, A. A. J. J. Z. Y. J. Nat. Genet. 1998; 20: PubMed Scopus Google Scholar, S. S.T. A. M. Maher Res. 2005; PubMed Scopus Google Scholar). that the target sequence can HDR pathways and by exogenous duplex and single strand modulation at frequencies 2–3 orders of magnitude those in experiments with the donors alone. 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A. 2003; PubMed Scopus Google Scholar, F. J.R. Mol. Cell. Biol. 2006; 26: PubMed Scopus Google Scholar). In that a in the frequency of SSO sequence conversion, was for SSO The that SSO repair of a break the annealing of a in which the SSO repair by annealing to a single strand of the a for The break would be in the In competition experiments asked whether the SSO would for ends that would interact with the duplex knock-in donor. We that the frequency of knock-in was unaffected by the of the SSO in experiments by the or would that the ends that the duplex knock-in donor were not to the SSO. would that to a pathway is by than the of nucleic acid that the for both homologous recombination repair and pathway is a end F. J.R. Mol. Cell. Biol. 2006; 26: PubMed Scopus Google Scholar), there would have to be a in the with the SSO to one the from the in S. to mammalian cells, the of a on a resected end, for strand would of that end with the SSO. the SSO would not for ends that the knock-in donor for a and of the of the SSO with intermediates in DSB repair be of to which factors the to ends by the SSO in mammalian cells. with

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