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Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction.

Olsen PA, Gelazauskaite M, Randøl M, Krauss S - BMC Mol. Biol. (2010)

Bottom Line: Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs.As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Cellular and Genetic Therapy, Institute of Microbiology, Oslo University Hospital, Rikshospitalet, Gausdadalleen 21, 0349 Oslo, Norway. petter.angell.olsen@rr-research.no

ABSTRACT

Background: Formation of site specific genomic double strand breaks (DSBs), induced by the expression of a pair of engineered zinc-finger nucleases (ZFNs), dramatically increases the rates of homologous recombination (HR) between a specific genomic target and a donor plasmid. However, for the safe use of ZFN induced HR in practical applications, possible adverse effects of the technology such as cytotoxicity and genotoxicity need to be well understood. In this work, off-target activity of a pair of ZFNs has been examined by measuring the ratio between HR and illegitimate genomic integration in cells that are growing exponentially, and in cells that have been arrested in the G2/M phase.

Results: A reporter cell line that contained consensus ZFN binding sites in an enhanced green fluorescent protein (EGFP) reporter gene was used to measure ratios between HR and non-homologous integration of a plasmid template. Both in human cells (HEK 293) containing the consensus ZFN binding sites and in cells lacking the ZFN binding sites, a 3.5 fold increase in the level of illegitimate integration was observed upon ZFN expression. Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs. Additionally, in a fraction of the ZFN treated cells the co-occurrence of both specific HR and illegitimate integration was observed. As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.

Conclusions: The demonstration that ZFN expression, in addition to stimulating specific gene targeting by HR, leads to increased rates of illegitimate integration emphasizes the importance of careful characterization of ZFN treated cells. In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.

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Effects of G2/M arrest of the target cells on gene correction and illegitimate integration. (A) Cell cycle profiles of exponentially growing, nocodazole arrested, and nocodazole arrested and subsequently released 293-Flp-mEGFP cells (left, middle and right panels, respectively). Histograms displaying the distribution of the DNA content of treated cells as measured by propidium iodide (PI) staining are shown. (B) Quantification of gene correction in exponentially growing (black bars) and G2/M arrested (gray bars) 293-Flp-mEGFP cells after transfection with pDonor, pZFN-L and pZFN-R as indicated. The amount of green cells was measured 72 hours after transfection by flow cytometry as in Fig. 2. (C) Quantification of illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). Cells were treated as in B and the level of illegitimate integration was quantified by measuring the number of puromycin resistant colonies formed as in Fig. 3B. The results presented are normalized for different PE in the untreated and G2/M arrested cells. (D) Presentation of the ratio between HR and illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). The ratio was calculated from the results in B and C.
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Figure 5: Effects of G2/M arrest of the target cells on gene correction and illegitimate integration. (A) Cell cycle profiles of exponentially growing, nocodazole arrested, and nocodazole arrested and subsequently released 293-Flp-mEGFP cells (left, middle and right panels, respectively). Histograms displaying the distribution of the DNA content of treated cells as measured by propidium iodide (PI) staining are shown. (B) Quantification of gene correction in exponentially growing (black bars) and G2/M arrested (gray bars) 293-Flp-mEGFP cells after transfection with pDonor, pZFN-L and pZFN-R as indicated. The amount of green cells was measured 72 hours after transfection by flow cytometry as in Fig. 2. (C) Quantification of illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). Cells were treated as in B and the level of illegitimate integration was quantified by measuring the number of puromycin resistant colonies formed as in Fig. 3B. The results presented are normalized for different PE in the untreated and G2/M arrested cells. (D) Presentation of the ratio between HR and illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). The ratio was calculated from the results in B and C.

Mentions: Cell cycle status is an important factor in the decision whether a genomic DSB is repaired by HR or NHEJ. While NHEJ is active throughout the cell cycle, HR is mainly active in the late S and G2/M phases [37]. To examine the influence of cell cycle phase on the levels of both HR and illegitimate integration, 293-Flp-mEGFP cells were arrested in the G2/M cell cycle phase by the microtubule-depolymerizing drug nocodazole, that reversibly arrests cells in the G2/M phase [38]. As seen in Fig. 5A, incubation of cells with nocodazole for 24 hours resulted in a clear accumulation of cells in the G2/M cell cycle phase (middle panel). After removal and further incubation for 48 hours without nocodazole, cells reentered normal proliferation (right panel) and displayed a cell cycle profile equivalent to untreated cells (left panel). When the percentage of ZFN induced green cells was compared between exponentially growing and in G2/M arrested 293-Flp-mEGFP cells, a 5.8 fold increase of HR was observed in the G2/M arrested cell (from 0.12% to 0.70%) (Fig. 5B). The observed differences in HR was not due to differences in the transfection efficiencies as transfection with a wt EGFP expression plasmid (pEGFP-c1) displayed similar frequencies both in untreated and nocodazole treated cells (not shown). Increased rates of HR following nocodazole induced G2/M cell cycle arrest are in agreement with Urnov et al. who observed increased levels (4.8 fold) of ZFN mediated HR in cells reversely arrested in the G2/M phase using vinblastine [8].


Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction.

Olsen PA, Gelazauskaite M, Randøl M, Krauss S - BMC Mol. Biol. (2010)

Effects of G2/M arrest of the target cells on gene correction and illegitimate integration. (A) Cell cycle profiles of exponentially growing, nocodazole arrested, and nocodazole arrested and subsequently released 293-Flp-mEGFP cells (left, middle and right panels, respectively). Histograms displaying the distribution of the DNA content of treated cells as measured by propidium iodide (PI) staining are shown. (B) Quantification of gene correction in exponentially growing (black bars) and G2/M arrested (gray bars) 293-Flp-mEGFP cells after transfection with pDonor, pZFN-L and pZFN-R as indicated. The amount of green cells was measured 72 hours after transfection by flow cytometry as in Fig. 2. (C) Quantification of illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). Cells were treated as in B and the level of illegitimate integration was quantified by measuring the number of puromycin resistant colonies formed as in Fig. 3B. The results presented are normalized for different PE in the untreated and G2/M arrested cells. (D) Presentation of the ratio between HR and illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). The ratio was calculated from the results in B and C.
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Figure 5: Effects of G2/M arrest of the target cells on gene correction and illegitimate integration. (A) Cell cycle profiles of exponentially growing, nocodazole arrested, and nocodazole arrested and subsequently released 293-Flp-mEGFP cells (left, middle and right panels, respectively). Histograms displaying the distribution of the DNA content of treated cells as measured by propidium iodide (PI) staining are shown. (B) Quantification of gene correction in exponentially growing (black bars) and G2/M arrested (gray bars) 293-Flp-mEGFP cells after transfection with pDonor, pZFN-L and pZFN-R as indicated. The amount of green cells was measured 72 hours after transfection by flow cytometry as in Fig. 2. (C) Quantification of illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). Cells were treated as in B and the level of illegitimate integration was quantified by measuring the number of puromycin resistant colonies formed as in Fig. 3B. The results presented are normalized for different PE in the untreated and G2/M arrested cells. (D) Presentation of the ratio between HR and illegitimate integration in exponentially growing (black bars) and G2/M arrested cells (grey bars). The ratio was calculated from the results in B and C.
Mentions: Cell cycle status is an important factor in the decision whether a genomic DSB is repaired by HR or NHEJ. While NHEJ is active throughout the cell cycle, HR is mainly active in the late S and G2/M phases [37]. To examine the influence of cell cycle phase on the levels of both HR and illegitimate integration, 293-Flp-mEGFP cells were arrested in the G2/M cell cycle phase by the microtubule-depolymerizing drug nocodazole, that reversibly arrests cells in the G2/M phase [38]. As seen in Fig. 5A, incubation of cells with nocodazole for 24 hours resulted in a clear accumulation of cells in the G2/M cell cycle phase (middle panel). After removal and further incubation for 48 hours without nocodazole, cells reentered normal proliferation (right panel) and displayed a cell cycle profile equivalent to untreated cells (left panel). When the percentage of ZFN induced green cells was compared between exponentially growing and in G2/M arrested 293-Flp-mEGFP cells, a 5.8 fold increase of HR was observed in the G2/M arrested cell (from 0.12% to 0.70%) (Fig. 5B). The observed differences in HR was not due to differences in the transfection efficiencies as transfection with a wt EGFP expression plasmid (pEGFP-c1) displayed similar frequencies both in untreated and nocodazole treated cells (not shown). Increased rates of HR following nocodazole induced G2/M cell cycle arrest are in agreement with Urnov et al. who observed increased levels (4.8 fold) of ZFN mediated HR in cells reversely arrested in the G2/M phase using vinblastine [8].

Bottom Line: Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs.As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section for Cellular and Genetic Therapy, Institute of Microbiology, Oslo University Hospital, Rikshospitalet, Gausdadalleen 21, 0349 Oslo, Norway. petter.angell.olsen@rr-research.no

ABSTRACT

Background: Formation of site specific genomic double strand breaks (DSBs), induced by the expression of a pair of engineered zinc-finger nucleases (ZFNs), dramatically increases the rates of homologous recombination (HR) between a specific genomic target and a donor plasmid. However, for the safe use of ZFN induced HR in practical applications, possible adverse effects of the technology such as cytotoxicity and genotoxicity need to be well understood. In this work, off-target activity of a pair of ZFNs has been examined by measuring the ratio between HR and illegitimate genomic integration in cells that are growing exponentially, and in cells that have been arrested in the G2/M phase.

Results: A reporter cell line that contained consensus ZFN binding sites in an enhanced green fluorescent protein (EGFP) reporter gene was used to measure ratios between HR and non-homologous integration of a plasmid template. Both in human cells (HEK 293) containing the consensus ZFN binding sites and in cells lacking the ZFN binding sites, a 3.5 fold increase in the level of illegitimate integration was observed upon ZFN expression. Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs. Additionally, in a fraction of the ZFN treated cells the co-occurrence of both specific HR and illegitimate integration was observed. As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.

Conclusions: The demonstration that ZFN expression, in addition to stimulating specific gene targeting by HR, leads to increased rates of illegitimate integration emphasizes the importance of careful characterization of ZFN treated cells. In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.

Show MeSH