Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of PTO in long-term survival of corrected cells.(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351460&req=5

pone-0036697-g006: Effects of PTO in long-term survival of corrected cells.(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.

Mentions: To further elucidate oligo design principles, we checked for long-term survival of EGFP+ cells two weeks after transfection while varying the number of PTO bonds 3′ to the mismatch (Figure 6a). This showed 3-5PTO bonds as optimal, resulting in at least ~10-fold increase in targeting efficiency compared to unmodified oligos. We also varied the position of the PTO bonds, the strand polarity of the oligo used and the presence of modified bases (Figure 6b). When using an oligo complementary to the transcribed strand (F5-31, -32) we were able to detect some EGFP+ cells, but only slightly above the control oligo F5-20, suggesting the strand bias against the transcribed strand observed in this cell line is not just due to the additional DNA replication necessary to express the corrected gene when targeting this strand. Comparing the presence of 3PTO bonds at the 3′ terminus (F5-30) vs. 3′ internal to the mismatch (F5-17) produced a ~4-fold reduction in EGFP+ cells, confirming that internal PTO modifications result in higher stable targeting frequencies. Comparing the natural (F5-29) and modified (F5-17) base oligos resulted in a similar 2-fold increase for the modified base as when assayed 48 hrs after transfection. Thus, optimizing the oligo design with modified base analogs and reducing the number of PTO bonds enabled us to generate stable, isogenic populations genetically modified with oligos.


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)

Effects of PTO in long-term survival of corrected cells.(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036697-g006: Effects of PTO in long-term survival of corrected cells.(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.
Mentions: To further elucidate oligo design principles, we checked for long-term survival of EGFP+ cells two weeks after transfection while varying the number of PTO bonds 3′ to the mismatch (Figure 6a). This showed 3-5PTO bonds as optimal, resulting in at least ~10-fold increase in targeting efficiency compared to unmodified oligos. We also varied the position of the PTO bonds, the strand polarity of the oligo used and the presence of modified bases (Figure 6b). When using an oligo complementary to the transcribed strand (F5-31, -32) we were able to detect some EGFP+ cells, but only slightly above the control oligo F5-20, suggesting the strand bias against the transcribed strand observed in this cell line is not just due to the additional DNA replication necessary to express the corrected gene when targeting this strand. Comparing the presence of 3PTO bonds at the 3′ terminus (F5-30) vs. 3′ internal to the mismatch (F5-17) produced a ~4-fold reduction in EGFP+ cells, confirming that internal PTO modifications result in higher stable targeting frequencies. Comparing the natural (F5-29) and modified (F5-17) base oligos resulted in a similar 2-fold increase for the modified base as when assayed 48 hrs after transfection. Thus, optimizing the oligo design with modified base analogs and reducing the number of PTO bonds enabled us to generate stable, isogenic populations genetically modified with oligos.

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