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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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Induction of immune-related genes in oligo-transfected cells.(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.
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pone-0036697-g007: Induction of immune-related genes in oligo-transfected cells.(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.

Mentions: To further explore this observation, we choose three highly differentially expressed genes known to be involved in antiviral/cellular immune responses: HLAB, IL32 and OAS3. We validated these genes by RT-qPCR, which allowed us to normalize the expression levels of corrected and uncorrected cells to untransfected cells(Fig. 7a). This revealed much higher induction levels for both populations relative to untransfected cells. For F5-17, the difference between transfected and untransfected cells was much higher (8–30 fold) than between EGFP+ and EGFP- cells (2–4 fold), suggesting that both non-corrected and corrected cells share an ‘oligo-transfected’ transcription profile. To verify that the gene changes were not due to EGFP itself, we checked the expression levels of EGFP+ and EGFP- cells after transfection with the F5-18 oligo. Like F5-17, F5-18 also generates EGFP+ cells, but F5-18 is a more toxic oligo as it contains three additional PTO bonds. If the upregulation of the three immune response genes we had observed were due to effects of the EGFP protein and not oligo toxicity, EGFP+ or EGFP- cells should have similar expression of these genes whether they had been transfected with F5-17 or F5-18. In contrast, however, the F5-18 transfected cells showed proportionally higher levels of induction of the three genes relative to F5-17, with EGFP+ cells still ~2–4 fold higher than EGFP- cells. One possible explanation for the difference between corrected and uncorrected cells would be that corrected cells got more oligos during transfection. We tested this by co-transfecting a targeting oligo along with a Cy5-labled one (F5-37). This showed that EGFP+ cells consistently had 2–5 times higher levels of oligos at varying oligo concentrations (Fig. 7b), suggesting that corrected cells may be proliferating less due to relatively higher oligo concentrations inducing a stronger immune response. Oligos with higher number of PTO modification have increased half-lives, which might lead to higher immune stimulation.


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)

Induction of immune-related genes in oligo-transfected cells.(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351460&req=5

pone-0036697-g007: Induction of immune-related genes in oligo-transfected cells.(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.
Mentions: To further explore this observation, we choose three highly differentially expressed genes known to be involved in antiviral/cellular immune responses: HLAB, IL32 and OAS3. We validated these genes by RT-qPCR, which allowed us to normalize the expression levels of corrected and uncorrected cells to untransfected cells(Fig. 7a). This revealed much higher induction levels for both populations relative to untransfected cells. For F5-17, the difference between transfected and untransfected cells was much higher (8–30 fold) than between EGFP+ and EGFP- cells (2–4 fold), suggesting that both non-corrected and corrected cells share an ‘oligo-transfected’ transcription profile. To verify that the gene changes were not due to EGFP itself, we checked the expression levels of EGFP+ and EGFP- cells after transfection with the F5-18 oligo. Like F5-17, F5-18 also generates EGFP+ cells, but F5-18 is a more toxic oligo as it contains three additional PTO bonds. If the upregulation of the three immune response genes we had observed were due to effects of the EGFP protein and not oligo toxicity, EGFP+ or EGFP- cells should have similar expression of these genes whether they had been transfected with F5-17 or F5-18. In contrast, however, the F5-18 transfected cells showed proportionally higher levels of induction of the three genes relative to F5-17, with EGFP+ cells still ~2–4 fold higher than EGFP- cells. One possible explanation for the difference between corrected and uncorrected cells would be that corrected cells got more oligos during transfection. We tested this by co-transfecting a targeting oligo along with a Cy5-labled one (F5-37). This showed that EGFP+ cells consistently had 2–5 times higher levels of oligos at varying oligo concentrations (Fig. 7b), suggesting that corrected cells may be proliferating less due to relatively higher oligo concentrations inducing a stronger immune response. Oligos with higher number of PTO modification have increased half-lives, which might lead to higher immune stimulation.

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