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p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

Herrmann F, Garriga-Canut M, Baumstark R, Fajardo-Sanchez E, Cotterell J, Minoche A, Himmelbauer H, Isalan M - PLoS ONE (2011)

Bottom Line: The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors.We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries.We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation and UPF, Barcelona, Spain.

ABSTRACT
The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

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Yeast one-hybrid based selection for ZFNs.(A) The ZF-target site is cloned upstream of a minimal promoter (Pmin) and the HIS3 reporter in the bait plasmid. Any interaction between a ZFP:Gal4-AD fusion protein and the target sequence stimulates transcription of HIS3 allowing selection on His-selective medium (B) The ZF-library:Gal4-AD fusion is generated by yeast recombination of a PCR-generated four-finger library cassette with the linearized prey plasmid; no extra library cloning step is required. Thus, bait plasmid, linearized prey plasmid and library PCR cassette are co-transformed into yeast. After incubation for 3–5 days, expression from the HIS3 reporter is detected in colonies that are able to grow on a selection medium that lacks histidine and contains 3-AT (see Methods). ZFP from positive clones are rescued by colony-PCR, are fused to a FokI-domain and are tested for activity by an in vitro cleavage assay. (C) Zinc finger library PCR template (z1166L). The template is based on 2×2-finger units from F2-F3 of the Zif268 sequence [65]. Each pair of 2-finger units is separated by a longer TGSERP linker [66]. The final linker, (QNKKQLVKSEL) is compatible with the FokI sequence and is adapted from [16]. DNA-recognition helices are selectively randomised at certain positions (marked “X”). Full sequences and randomisation strategy are in Methods S1.
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pone-0020913-g002: Yeast one-hybrid based selection for ZFNs.(A) The ZF-target site is cloned upstream of a minimal promoter (Pmin) and the HIS3 reporter in the bait plasmid. Any interaction between a ZFP:Gal4-AD fusion protein and the target sequence stimulates transcription of HIS3 allowing selection on His-selective medium (B) The ZF-library:Gal4-AD fusion is generated by yeast recombination of a PCR-generated four-finger library cassette with the linearized prey plasmid; no extra library cloning step is required. Thus, bait plasmid, linearized prey plasmid and library PCR cassette are co-transformed into yeast. After incubation for 3–5 days, expression from the HIS3 reporter is detected in colonies that are able to grow on a selection medium that lacks histidine and contains 3-AT (see Methods). ZFP from positive clones are rescued by colony-PCR, are fused to a FokI-domain and are tested for activity by an in vitro cleavage assay. (C) Zinc finger library PCR template (z1166L). The template is based on 2×2-finger units from F2-F3 of the Zif268 sequence [65]. Each pair of 2-finger units is separated by a longer TGSERP linker [66]. The final linker, (QNKKQLVKSEL) is compatible with the FokI sequence and is adapted from [16]. DNA-recognition helices are selectively randomised at certain positions (marked “X”). Full sequences and randomisation strategy are in Methods S1.

Mentions: One-hybrid screening in yeast is a powerful method to rapidly identify DNA-binding peptides that can interact with a specific DNA sequence of interest. We therefore developed a yeast one-hybrid (Y1H) selection system for zinc finger peptides, based on the commercially-available Matchmaker Kit (Clontech). The system allowed us to construct semi-randomised zinc finger libraries by PCR (without cloning), and to screen them in one step, by yeast transformation and plating on selective medium (Fig. 2).


p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

Herrmann F, Garriga-Canut M, Baumstark R, Fajardo-Sanchez E, Cotterell J, Minoche A, Himmelbauer H, Isalan M - PLoS ONE (2011)

Yeast one-hybrid based selection for ZFNs.(A) The ZF-target site is cloned upstream of a minimal promoter (Pmin) and the HIS3 reporter in the bait plasmid. Any interaction between a ZFP:Gal4-AD fusion protein and the target sequence stimulates transcription of HIS3 allowing selection on His-selective medium (B) The ZF-library:Gal4-AD fusion is generated by yeast recombination of a PCR-generated four-finger library cassette with the linearized prey plasmid; no extra library cloning step is required. Thus, bait plasmid, linearized prey plasmid and library PCR cassette are co-transformed into yeast. After incubation for 3–5 days, expression from the HIS3 reporter is detected in colonies that are able to grow on a selection medium that lacks histidine and contains 3-AT (see Methods). ZFP from positive clones are rescued by colony-PCR, are fused to a FokI-domain and are tested for activity by an in vitro cleavage assay. (C) Zinc finger library PCR template (z1166L). The template is based on 2×2-finger units from F2-F3 of the Zif268 sequence [65]. Each pair of 2-finger units is separated by a longer TGSERP linker [66]. The final linker, (QNKKQLVKSEL) is compatible with the FokI sequence and is adapted from [16]. DNA-recognition helices are selectively randomised at certain positions (marked “X”). Full sequences and randomisation strategy are in Methods S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111460&req=5

pone-0020913-g002: Yeast one-hybrid based selection for ZFNs.(A) The ZF-target site is cloned upstream of a minimal promoter (Pmin) and the HIS3 reporter in the bait plasmid. Any interaction between a ZFP:Gal4-AD fusion protein and the target sequence stimulates transcription of HIS3 allowing selection on His-selective medium (B) The ZF-library:Gal4-AD fusion is generated by yeast recombination of a PCR-generated four-finger library cassette with the linearized prey plasmid; no extra library cloning step is required. Thus, bait plasmid, linearized prey plasmid and library PCR cassette are co-transformed into yeast. After incubation for 3–5 days, expression from the HIS3 reporter is detected in colonies that are able to grow on a selection medium that lacks histidine and contains 3-AT (see Methods). ZFP from positive clones are rescued by colony-PCR, are fused to a FokI-domain and are tested for activity by an in vitro cleavage assay. (C) Zinc finger library PCR template (z1166L). The template is based on 2×2-finger units from F2-F3 of the Zif268 sequence [65]. Each pair of 2-finger units is separated by a longer TGSERP linker [66]. The final linker, (QNKKQLVKSEL) is compatible with the FokI sequence and is adapted from [16]. DNA-recognition helices are selectively randomised at certain positions (marked “X”). Full sequences and randomisation strategy are in Methods S1.
Mentions: One-hybrid screening in yeast is a powerful method to rapidly identify DNA-binding peptides that can interact with a specific DNA sequence of interest. We therefore developed a yeast one-hybrid (Y1H) selection system for zinc finger peptides, based on the commercially-available Matchmaker Kit (Clontech). The system allowed us to construct semi-randomised zinc finger libraries by PCR (without cloning), and to screen them in one step, by yeast transformation and plating on selective medium (Fig. 2).

Bottom Line: The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors.We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries.We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'.

View Article: PubMed Central - PubMed

Affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation and UPF, Barcelona, Spain.

ABSTRACT
The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

Show MeSH
Related in: MedlinePlus