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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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Analysis of the PuroR genomic integration site in 293-Flp-mEGFP cells. (A) The integrity of the mEGFP gene in non-green puromycin resistant colonies of 293-Flp-mEGFP cells was analyzed after transfection with pDonor together with the ZFNs. Genomic DNA isolated from 25 individual colonies was subjected to PCR using primer pairs as indicated above the panels. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers in opposite orientations (Pu-1 and Pu-2). Control PCR's (rightmost gel, ctr) using pEGFP-BABE-puro as template was performed with the primer pairs: P-amp + Pu-1 (lane 1), P1 + Pu-1 (lane 2), P1 + Pu-1 without template (lane 3) and PCR amplification produced the anticipated amplification products (2364 bp, 1429 bp and no product) verifying that primers worked under the given conditions. (B) PCR analysis of genomic DNA from 26 individual colonies displaying both green fluorescence (HR) and puromycin resistance (illegitimate recombination) after transfection with the pDonor and ZFNs. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers (Pu-1 and Pu-2) to investigate if the ΔEGFP and PuroR cassette had integrated in the genome of the 293-Flp-mEGFP cells as a single fragment or not. M represents the size marker and sizes of relevant bands are specified. Position of the PCR reactions from the individual colonies is indicated below the lanes.
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Figure 4: Analysis of the PuroR genomic integration site in 293-Flp-mEGFP cells. (A) The integrity of the mEGFP gene in non-green puromycin resistant colonies of 293-Flp-mEGFP cells was analyzed after transfection with pDonor together with the ZFNs. Genomic DNA isolated from 25 individual colonies was subjected to PCR using primer pairs as indicated above the panels. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers in opposite orientations (Pu-1 and Pu-2). Control PCR's (rightmost gel, ctr) using pEGFP-BABE-puro as template was performed with the primer pairs: P-amp + Pu-1 (lane 1), P1 + Pu-1 (lane 2), P1 + Pu-1 without template (lane 3) and PCR amplification produced the anticipated amplification products (2364 bp, 1429 bp and no product) verifying that primers worked under the given conditions. (B) PCR analysis of genomic DNA from 26 individual colonies displaying both green fluorescence (HR) and puromycin resistance (illegitimate recombination) after transfection with the pDonor and ZFNs. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers (Pu-1 and Pu-2) to investigate if the ΔEGFP and PuroR cassette had integrated in the genome of the 293-Flp-mEGFP cells as a single fragment or not. M represents the size marker and sizes of relevant bands are specified. Position of the PCR reactions from the individual colonies is indicated below the lanes.

Mentions: To investigate whether the observed increase rates of illegitimate integration after ZFN exposure was due to an integration of the PuroR cassette into the genomic mEGFP gene, PCR analysis of the genomic DNA from 25 puromycin resistant colonies was performed. In two separate PCR analyses using the same forward primer (P1 in Fig. 1B), specific to the first bases in the genomic mEGFP gene, and primers specific for the PuroR gene either in forward or reverse orientation (Pu-1 or Pu-2 in Fig. 1B), no amplification products were obtained (Fig. 4A). In a positive control PCR using a plasmid template, the functionality of the primers and PCR conditions used were verified (Fig. 4A ctr). Accordingly, the failure to produce an amplification product from the genomic DNA was most likely due to the absence of the PuroR sequence in the mEGFP gene. To further test whether the genomic mEGFP in the puromycin resistant colonies was intact, PCR analysis of the puromycin resistant colonies with primers flanking the mEGFP gene (P2 and P3 in Fig 1B) was carried out. PCR analysis of the puromycin resistant colonies with primers P2 and P3 produced an amplification product of approximately 600 bp corresponding to the expected size of an uninterrupted mEGFP gene (610 bp) in all the 25 colonies (not shown). Taken together these results established that the increased rates of illegitimate integration following ZFN expression was not due to genomic integration of the PuroR cassette in the genomic mEGFP site where the ZFNs were designed to form a DSB. Given that that the average occurrence of the canonical 18 bp ZFN recognition sequence statistically is only one in the mammalian genome, the observed increased rates of illegitimate integration following ZFN expression was most likely due to formation off-target genomic DSBs by the ZFNs.


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)

Analysis of the PuroR genomic integration site in 293-Flp-mEGFP cells. (A) The integrity of the mEGFP gene in non-green puromycin resistant colonies of 293-Flp-mEGFP cells was analyzed after transfection with pDonor together with the ZFNs. Genomic DNA isolated from 25 individual colonies was subjected to PCR using primer pairs as indicated above the panels. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers in opposite orientations (Pu-1 and Pu-2). Control PCR's (rightmost gel, ctr) using pEGFP-BABE-puro as template was performed with the primer pairs: P-amp + Pu-1 (lane 1), P1 + Pu-1 (lane 2), P1 + Pu-1 without template (lane 3) and PCR amplification produced the anticipated amplification products (2364 bp, 1429 bp and no product) verifying that primers worked under the given conditions. (B) PCR analysis of genomic DNA from 26 individual colonies displaying both green fluorescence (HR) and puromycin resistance (illegitimate recombination) after transfection with the pDonor and ZFNs. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers (Pu-1 and Pu-2) to investigate if the ΔEGFP and PuroR cassette had integrated in the genome of the 293-Flp-mEGFP cells as a single fragment or not. M represents the size marker and sizes of relevant bands are specified. Position of the PCR reactions from the individual colonies is indicated below the lanes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Analysis of the PuroR genomic integration site in 293-Flp-mEGFP cells. (A) The integrity of the mEGFP gene in non-green puromycin resistant colonies of 293-Flp-mEGFP cells was analyzed after transfection with pDonor together with the ZFNs. Genomic DNA isolated from 25 individual colonies was subjected to PCR using primer pairs as indicated above the panels. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers in opposite orientations (Pu-1 and Pu-2). Control PCR's (rightmost gel, ctr) using pEGFP-BABE-puro as template was performed with the primer pairs: P-amp + Pu-1 (lane 1), P1 + Pu-1 (lane 2), P1 + Pu-1 without template (lane 3) and PCR amplification produced the anticipated amplification products (2364 bp, 1429 bp and no product) verifying that primers worked under the given conditions. (B) PCR analysis of genomic DNA from 26 individual colonies displaying both green fluorescence (HR) and puromycin resistance (illegitimate recombination) after transfection with the pDonor and ZFNs. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers (Pu-1 and Pu-2) to investigate if the ΔEGFP and PuroR cassette had integrated in the genome of the 293-Flp-mEGFP cells as a single fragment or not. M represents the size marker and sizes of relevant bands are specified. Position of the PCR reactions from the individual colonies is indicated below the lanes.
Mentions: To investigate whether the observed increase rates of illegitimate integration after ZFN exposure was due to an integration of the PuroR cassette into the genomic mEGFP gene, PCR analysis of the genomic DNA from 25 puromycin resistant colonies was performed. In two separate PCR analyses using the same forward primer (P1 in Fig. 1B), specific to the first bases in the genomic mEGFP gene, and primers specific for the PuroR gene either in forward or reverse orientation (Pu-1 or Pu-2 in Fig. 1B), no amplification products were obtained (Fig. 4A). In a positive control PCR using a plasmid template, the functionality of the primers and PCR conditions used were verified (Fig. 4A ctr). Accordingly, the failure to produce an amplification product from the genomic DNA was most likely due to the absence of the PuroR sequence in the mEGFP gene. To further test whether the genomic mEGFP in the puromycin resistant colonies was intact, PCR analysis of the puromycin resistant colonies with primers flanking the mEGFP gene (P2 and P3 in Fig 1B) was carried out. PCR analysis of the puromycin resistant colonies with primers P2 and P3 produced an amplification product of approximately 600 bp corresponding to the expected size of an uninterrupted mEGFP gene (610 bp) in all the 25 colonies (not shown). Taken together these results established that the increased rates of illegitimate integration following ZFN expression was not due to genomic integration of the PuroR cassette in the genomic mEGFP site where the ZFNs were designed to form a DSB. Given that that the average occurrence of the canonical 18 bp ZFN recognition sequence statistically is only one in the mammalian genome, the observed increased rates of illegitimate integration following ZFN expression was most likely due to formation off-target genomic DSBs by the ZFNs.

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