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Stable gene targeting in human cells using single-strand oligonucleotides with modified bases.

Rios X, Briggs AW, Christodoulou D, Gorham JM, Seidman JG, Church GM - PLoS ONE (2012)

Bottom Line: Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds.We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells.Further optimization of this method should allow rapid and scalable genome engineering in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.

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Proliferation defect in corrected cells.Proliferation is inversely proportional to the MFI of the CellTrace Violet dye. Cells were treated with CellTrace Violet dye, then split and transfected with oligos 48 hrs later. Cells were fixed at each time point and run together on a LSRfortessa cell analyzer at the end of the time course (a) Flow cytometry dot plots showing relative proliferation of EGFP+ and EGFP- cells generated with different targeting oligos, at four different time points post-transfection. %EGFP shown as average±s.d. n = 6. (b) Proliferation index is calculated as the inverse MFI of CellTrace ×106 for each population. (c) Cells treated with anti-microtubule drugs Taxol and Vincristine 24 h after transfection with oligo F5-17 and 24 h exposure time, checked at 48 h and 96 h post-transfection. n = 4.
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pone-0036697-g004: Proliferation defect in corrected cells.Proliferation is inversely proportional to the MFI of the CellTrace Violet dye. Cells were treated with CellTrace Violet dye, then split and transfected with oligos 48 hrs later. Cells were fixed at each time point and run together on a LSRfortessa cell analyzer at the end of the time course (a) Flow cytometry dot plots showing relative proliferation of EGFP+ and EGFP- cells generated with different targeting oligos, at four different time points post-transfection. %EGFP shown as average±s.d. n = 6. (b) Proliferation index is calculated as the inverse MFI of CellTrace ×106 for each population. (c) Cells treated with anti-microtubule drugs Taxol and Vincristine 24 h after transfection with oligo F5-17 and 24 h exposure time, checked at 48 h and 96 h post-transfection. n = 4.

Mentions: We hypothesized that reducing the number of PTO bonds might increase the number of proliferating EGFP+ cells. However, since reducing the number of PTO bonds also decreases targeting efficiency, we decided to concentrate on protecting the 3′ end. Furthermore, we decided to test internally protected oligos, since these have been found to lead to higher survival rates [20]. We designed two new oligos with three or six PTO bonds 3′ to the centrally located mismatch, all using a FU base analog (Table 1, Fig. 3b). Previous work has described the defect in oligo-corrected cells as a G2/M cell cycle arrest [20]–[21], [24], with few cells moving past it. In order to generate a more detailed view of the effects of oligos on proliferation, we tracked cells using the CellTrace Violet dye [31]–[32] (Figure 4a). This allowed us to follow the relative proliferation rate of the corrected (EGFP+) and uncorrected (EGFP-) cell populations by flow cytometry as the inverse dilution rate of the CellTrace dye’s Mean Fluorescent Intensity (MFI) (Figure 4b). First, we observe that in general EGFP+ cells proliferate slower than corresponding EGFP- cells. Second, the proliferation of EGFP+ cells inversely correlates with the number of PTO bonds of the oligo used to generate them, with the EGFP+ cells from the F5-8 (12 PTO) oligo hardly proliferating and the F5-18(6 PTO) having an intermediate phenotype. The proliferation rate of corrected cells depended on the number of PTO bonds on the oligo, suggesting it is the correcting oligo itself and not the expression of EGFP the cause of their proliferation defect. To functionally validate this observation, we treated F5-17 transfected cells with anti-microtubule drugs, which are preferentially toxic to proliferating cells [33], 24 hrs post-transfection (Fig. 4c). This produced an increase of EGFP+ cells at 48 and 96 hrs, suggesting that non-corrected cells had been preferentially inhibited, and therefore that corrected cells experience delayed proliferation. To verify that the effects of the anti-microtubule drugs were not due to endosome destabilization leading to increased oligo release, cell were treated with chloroquine, which actually slightly decreased targeting frequencies (not shown).


Stable gene targeting in human cells using single-strand oligonucleotides with modified bases.

Rios X, Briggs AW, Christodoulou D, Gorham JM, Seidman JG, Church GM - PLoS ONE (2012)

Proliferation defect in corrected cells.Proliferation is inversely proportional to the MFI of the CellTrace Violet dye. Cells were treated with CellTrace Violet dye, then split and transfected with oligos 48 hrs later. Cells were fixed at each time point and run together on a LSRfortessa cell analyzer at the end of the time course (a) Flow cytometry dot plots showing relative proliferation of EGFP+ and EGFP- cells generated with different targeting oligos, at four different time points post-transfection. %EGFP shown as average±s.d. n = 6. (b) Proliferation index is calculated as the inverse MFI of CellTrace ×106 for each population. (c) Cells treated with anti-microtubule drugs Taxol and Vincristine 24 h after transfection with oligo F5-17 and 24 h exposure time, checked at 48 h and 96 h post-transfection. n = 4.
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Related In: Results  -  Collection

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pone-0036697-g004: Proliferation defect in corrected cells.Proliferation is inversely proportional to the MFI of the CellTrace Violet dye. Cells were treated with CellTrace Violet dye, then split and transfected with oligos 48 hrs later. Cells were fixed at each time point and run together on a LSRfortessa cell analyzer at the end of the time course (a) Flow cytometry dot plots showing relative proliferation of EGFP+ and EGFP- cells generated with different targeting oligos, at four different time points post-transfection. %EGFP shown as average±s.d. n = 6. (b) Proliferation index is calculated as the inverse MFI of CellTrace ×106 for each population. (c) Cells treated with anti-microtubule drugs Taxol and Vincristine 24 h after transfection with oligo F5-17 and 24 h exposure time, checked at 48 h and 96 h post-transfection. n = 4.
Mentions: We hypothesized that reducing the number of PTO bonds might increase the number of proliferating EGFP+ cells. However, since reducing the number of PTO bonds also decreases targeting efficiency, we decided to concentrate on protecting the 3′ end. Furthermore, we decided to test internally protected oligos, since these have been found to lead to higher survival rates [20]. We designed two new oligos with three or six PTO bonds 3′ to the centrally located mismatch, all using a FU base analog (Table 1, Fig. 3b). Previous work has described the defect in oligo-corrected cells as a G2/M cell cycle arrest [20]–[21], [24], with few cells moving past it. In order to generate a more detailed view of the effects of oligos on proliferation, we tracked cells using the CellTrace Violet dye [31]–[32] (Figure 4a). This allowed us to follow the relative proliferation rate of the corrected (EGFP+) and uncorrected (EGFP-) cell populations by flow cytometry as the inverse dilution rate of the CellTrace dye’s Mean Fluorescent Intensity (MFI) (Figure 4b). First, we observe that in general EGFP+ cells proliferate slower than corresponding EGFP- cells. Second, the proliferation of EGFP+ cells inversely correlates with the number of PTO bonds of the oligo used to generate them, with the EGFP+ cells from the F5-8 (12 PTO) oligo hardly proliferating and the F5-18(6 PTO) having an intermediate phenotype. The proliferation rate of corrected cells depended on the number of PTO bonds on the oligo, suggesting it is the correcting oligo itself and not the expression of EGFP the cause of their proliferation defect. To functionally validate this observation, we treated F5-17 transfected cells with anti-microtubule drugs, which are preferentially toxic to proliferating cells [33], 24 hrs post-transfection (Fig. 4c). This produced an increase of EGFP+ cells at 48 and 96 hrs, suggesting that non-corrected cells had been preferentially inhibited, and therefore that corrected cells experience delayed proliferation. To verify that the effects of the anti-microtubule drugs were not due to endosome destabilization leading to increased oligo release, cell were treated with chloroquine, which actually slightly decreased targeting frequencies (not shown).

Bottom Line: Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds.We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells.Further optimization of this method should allow rapid and scalable genome engineering in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.

Show MeSH
Related in: MedlinePlus