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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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Varying PTOs in targeting oligos.All oligos in this figure are complementary to the non-transcribed strand’s first potential start codon TTG, similar to F5-3 in Fig. 1. (a) Oligo toxicity and targeting efficiency as a function of PTO modifications. PTO bond position shown in gray. ‘Total alive cells’ was estimated as the total gated PI- cells in 200 uL from a 24-well plate 48 hrs post oligo transfection. (b) Correction efficiencies of new oligo designs with reduced PTO bonds and using FU modified base. n = 4.
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pone-0036697-g003: Varying PTOs in targeting oligos.All oligos in this figure are complementary to the non-transcribed strand’s first potential start codon TTG, similar to F5-3 in Fig. 1. (a) Oligo toxicity and targeting efficiency as a function of PTO modifications. PTO bond position shown in gray. ‘Total alive cells’ was estimated as the total gated PI- cells in 200 uL from a 24-well plate 48 hrs post oligo transfection. (b) Correction efficiencies of new oligo designs with reduced PTO bonds and using FU modified base. n = 4.

Mentions: PTO modifications are used to protect oligos from nuclease degradation, however this modification has been reported to be toxic to cells [30]. To quantify this toxicity in relationship to mEGFP targeting efficiency we varied the number of PTO modifications in the targeting oligos and measured the effects on both cell survival and correction efficiency after 48 hrs. We found a clear trend where increased number of PTO bonds results in lower survival of cells 48 hrs after oligo transfection (Figure 3a), consistent with PTOs being toxic. In addition, an oligo with PTOs only at the 3′ end (F5-12) leads to higher correction efficiency than one with PTOs only at the 5′ end (F5-11, Fig. 3a). Interestingly, mEGFP correction steadily decreased with additional PTOs when there were more than six at each end. These results suggested that finding a balance between toxicity and efficiency might be necessary for generating stably corrected cells.


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)

Varying PTOs in targeting oligos.All oligos in this figure are complementary to the non-transcribed strand’s first potential start codon TTG, similar to F5-3 in Fig. 1. (a) Oligo toxicity and targeting efficiency as a function of PTO modifications. PTO bond position shown in gray. ‘Total alive cells’ was estimated as the total gated PI- cells in 200 uL from a 24-well plate 48 hrs post oligo transfection. (b) Correction efficiencies of new oligo designs with reduced PTO bonds and using FU modified base. n = 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351460&req=5

pone-0036697-g003: Varying PTOs in targeting oligos.All oligos in this figure are complementary to the non-transcribed strand’s first potential start codon TTG, similar to F5-3 in Fig. 1. (a) Oligo toxicity and targeting efficiency as a function of PTO modifications. PTO bond position shown in gray. ‘Total alive cells’ was estimated as the total gated PI- cells in 200 uL from a 24-well plate 48 hrs post oligo transfection. (b) Correction efficiencies of new oligo designs with reduced PTO bonds and using FU modified base. n = 4.
Mentions: PTO modifications are used to protect oligos from nuclease degradation, however this modification has been reported to be toxic to cells [30]. To quantify this toxicity in relationship to mEGFP targeting efficiency we varied the number of PTO modifications in the targeting oligos and measured the effects on both cell survival and correction efficiency after 48 hrs. We found a clear trend where increased number of PTO bonds results in lower survival of cells 48 hrs after oligo transfection (Figure 3a), consistent with PTOs being toxic. In addition, an oligo with PTOs only at the 3′ end (F5-12) leads to higher correction efficiency than one with PTOs only at the 5′ end (F5-11, Fig. 3a). Interestingly, mEGFP correction steadily decreased with additional PTOs when there were more than six at each end. These results suggested that finding a balance between toxicity and efficiency might be necessary for generating stably corrected cells.

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