Targeted gene knock in and sequence modulation mediated by a psoralen-linked triplex-forming oligonucleotide

Abstract

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.

FIGURE 1.

The sequences of the triplex target in the CHO Hprt gene and the triple helix-forming oligonucleotide linked to psoralen. The XbaI site is in italics, and the thymidine residues linked by the psoralen cross-link are in larger font.

FIGURE 2.

Targeted knock in of a duplex donor by the pso-TFO. a, schematic of the chromosomal target with the TFO-targeted cross-link site and the replacement duplex donor. The regions of homology are not to scale. Targeted knock in of the donor produces a cell that is Hprt mutant and resistant to neomycin. b, Southern blot analysis of individual clones with targeted knock in of the duplex donor. The Hprt gene is single copy in CHO cells, and precise knock in results in the shift of the restriction fragment from the size denoted by the light arrow (the control sample, rightmost lane) to that indicated by the heavy arrow.

FIGURE 3.

Influence of repair deficiency on pso-TFO-targeted knock in. Knock in of the duplex replacement donor was measured in wild type cells or cells deficient in XPD or ERCC1. The results are presented as the mean and S.D. of three or more independent experiments.

FIGURE 4.

Modification of the pso-TFO target site by SSO. a, sequence of the target and SSO. The target region in the AM12 cell line is shown with the sequence changes in italicized and enlarged font. The SSO donor restores the wild type sequence in the vicinity of the targeted cross-link and also introduces a C (in bold) in place of a T in the triplex target sequence. b, influence of SSO length and complementarity on donor activity at the pso-TFO target site. The designations Pu or Py reflect the strand containing either the purine or pyrimidine run in the triplex target. TFO^– refers to the activity of the SSO in the absence of the pso-TFO. Error bars represent S.D.

FIGURE 5.

Modulation of pso-TFO-targeted mutagenesis by wild type or mutant SSO donors. a, sequence of the mutant SSO donor TGRC-1 and the target region and product. The sequence of the wild type donor is shown in Fig. 4a. b, frequency of mutagenesis of the pso-TFO target with the TGRC-1 or wild type donor SSO. The open portion of the column for AE-07/TGRC-1 represents the frequency of TGRC-1-directed mutagenesis. Error bars represent the mean and S.D. of three or more independent experiments.

FIGURE 6.

Influence of repair deficiency on targeted mutagenesis by the TGRC-1 donor. Error bars represent the mean and S.D. of three or more independent experiments.

FIGURE 7.

The SSO does not influence knock in of the duplex donor by the pso-TFO. Cells were electroporated with the pso-TFO and the AM200 duplex donor with or without the SSO. Error bars represent the mean and S.D. of three or more independent experiments.

FIGURE 8.

Sequence modulation of an I-SceI site. a, targeted introduction of the I-SceI recognition sequence. The sequence of the donor SSO containing the I-SceI recognition sequence, the AM12 target sequence, and the sequence of the target region in the product cell line with the I-SceI recognition sequence adjacent to the triplex target. Also shown is the sequence of the competition donor SSO co-electroporated with the plasmid encoding I-SceI. b, sequence conversion by the SSO introduced into cells treated with the pso-TFO/UVA or the I-SceI expression plasmid. c, knock in of the AM200 donor in the presence or absence of the SSO in cells transfected with the I-SceI expression plasmid. Error bars represent the mean and S.D. of three or more independent experiments.