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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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Solexa-Illumina deep sequencing of ZFN-treated cells.(A) Short 31bp reads detect ZFN-induced non-homologous end-joining (NHEJ) events in HEK293T cells. The FokI cutting region (CAACTA) is indicated in bold, deletions are shown with “−” and insertions are underlined. (B) 104bp-read protocol: quantifying the rate of insertions and deletions induced by ZFN under a PGK promoter. SF268, K562 and BT549 cells were kept at 37°C or subjected to transient cold shock to increase NHEJ (30°C; [63]). ZFN plasmid amounts (0, 1 and 1.5 µg) are indicated by −, + asnd ++, respectively. Obligate heterodimer FokI mutants [57] are indicated where used (ObH). (C) Controls using wt∶mutant plasmids at ratios of 100∶1 and 1000∶1. After mixing, samples were treated identically as for other Solexa samples (3bp-barcode PCR, adapter ligation etc.). The proportion of wt∶mutant sequence in the Solexa output was then calculated. (D) Quantifying the rate of insertion of a barcoded (wt coding sequence) donor plasmid into the genomic p53 locus, in SF268 cells. CMV and PGK promoters were tested in combination with wt or ObH FokI nuclease domains. (E) A similar gene repair experiment in BT549 cells. Genomic DNA was collected 7 days after ZFN and donor plasmid transfection, to reduce background.
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pone-0020913-g006: Solexa-Illumina deep sequencing of ZFN-treated cells.(A) Short 31bp reads detect ZFN-induced non-homologous end-joining (NHEJ) events in HEK293T cells. The FokI cutting region (CAACTA) is indicated in bold, deletions are shown with “−” and insertions are underlined. (B) 104bp-read protocol: quantifying the rate of insertions and deletions induced by ZFN under a PGK promoter. SF268, K562 and BT549 cells were kept at 37°C or subjected to transient cold shock to increase NHEJ (30°C; [63]). ZFN plasmid amounts (0, 1 and 1.5 µg) are indicated by −, + asnd ++, respectively. Obligate heterodimer FokI mutants [57] are indicated where used (ObH). (C) Controls using wt∶mutant plasmids at ratios of 100∶1 and 1000∶1. After mixing, samples were treated identically as for other Solexa samples (3bp-barcode PCR, adapter ligation etc.). The proportion of wt∶mutant sequence in the Solexa output was then calculated. (D) Quantifying the rate of insertion of a barcoded (wt coding sequence) donor plasmid into the genomic p53 locus, in SF268 cells. CMV and PGK promoters were tested in combination with wt or ObH FokI nuclease domains. (E) A similar gene repair experiment in BT549 cells. Genomic DNA was collected 7 days after ZFN and donor plasmid transfection, to reduce background.

Mentions: First, using 31bp reads, we observed NHEJ from ZFN in HEK293T cells (Fig. 6A). The method involved 2 rounds of PCR: one external genomic PCR and one internal PCR to introduce 3bp sequencing barcodes and an MmeI cleavage site (see Methods S1). MmeI digestion allowed sequencing-adapter ligation as close to the region of interest as possible but, as a result, the method was qualitative rather than quantitative. Nonetheless, the method showed that ZFN treatment was required to observe insertions and deletions around the genomic cutting site; these mutations can be useful for knocking out genes [38], [39].


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)

Solexa-Illumina deep sequencing of ZFN-treated cells.(A) Short 31bp reads detect ZFN-induced non-homologous end-joining (NHEJ) events in HEK293T cells. The FokI cutting region (CAACTA) is indicated in bold, deletions are shown with “−” and insertions are underlined. (B) 104bp-read protocol: quantifying the rate of insertions and deletions induced by ZFN under a PGK promoter. SF268, K562 and BT549 cells were kept at 37°C or subjected to transient cold shock to increase NHEJ (30°C; [63]). ZFN plasmid amounts (0, 1 and 1.5 µg) are indicated by −, + asnd ++, respectively. Obligate heterodimer FokI mutants [57] are indicated where used (ObH). (C) Controls using wt∶mutant plasmids at ratios of 100∶1 and 1000∶1. After mixing, samples were treated identically as for other Solexa samples (3bp-barcode PCR, adapter ligation etc.). The proportion of wt∶mutant sequence in the Solexa output was then calculated. (D) Quantifying the rate of insertion of a barcoded (wt coding sequence) donor plasmid into the genomic p53 locus, in SF268 cells. CMV and PGK promoters were tested in combination with wt or ObH FokI nuclease domains. (E) A similar gene repair experiment in BT549 cells. Genomic DNA was collected 7 days after ZFN and donor plasmid transfection, to reduce background.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111460&req=5

pone-0020913-g006: Solexa-Illumina deep sequencing of ZFN-treated cells.(A) Short 31bp reads detect ZFN-induced non-homologous end-joining (NHEJ) events in HEK293T cells. The FokI cutting region (CAACTA) is indicated in bold, deletions are shown with “−” and insertions are underlined. (B) 104bp-read protocol: quantifying the rate of insertions and deletions induced by ZFN under a PGK promoter. SF268, K562 and BT549 cells were kept at 37°C or subjected to transient cold shock to increase NHEJ (30°C; [63]). ZFN plasmid amounts (0, 1 and 1.5 µg) are indicated by −, + asnd ++, respectively. Obligate heterodimer FokI mutants [57] are indicated where used (ObH). (C) Controls using wt∶mutant plasmids at ratios of 100∶1 and 1000∶1. After mixing, samples were treated identically as for other Solexa samples (3bp-barcode PCR, adapter ligation etc.). The proportion of wt∶mutant sequence in the Solexa output was then calculated. (D) Quantifying the rate of insertion of a barcoded (wt coding sequence) donor plasmid into the genomic p53 locus, in SF268 cells. CMV and PGK promoters were tested in combination with wt or ObH FokI nuclease domains. (E) A similar gene repair experiment in BT549 cells. Genomic DNA was collected 7 days after ZFN and donor plasmid transfection, to reduce background.
Mentions: First, using 31bp reads, we observed NHEJ from ZFN in HEK293T cells (Fig. 6A). The method involved 2 rounds of PCR: one external genomic PCR and one internal PCR to introduce 3bp sequencing barcodes and an MmeI cleavage site (see Methods S1). MmeI digestion allowed sequencing-adapter ligation as close to the region of interest as possible but, as a result, the method was qualitative rather than quantitative. Nonetheless, the method showed that ZFN treatment was required to observe insertions and deletions around the genomic cutting site; these mutations can be useful for knocking out genes [38], [39].

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