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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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Related in: MedlinePlus

Generating double mutants.Oligos complementary to the first start codon in the non-transcribed strand and containing a second mismatch, either an insertion (∧A) or substitution (-A), with silenced MSH2 and assayed 36 hrs after transfection. Targeting oligos shown in duplex 3′-’to 5′-, with PTO highlighted in gray and mismatches shown with an asterisk. F5-20 was used as non-correcting control. n = 4.
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pone-0036697-g005: Generating double mutants.Oligos complementary to the first start codon in the non-transcribed strand and containing a second mismatch, either an insertion (∧A) or substitution (-A), with silenced MSH2 and assayed 36 hrs after transfection. Targeting oligos shown in duplex 3′-’to 5′-, with PTO highlighted in gray and mismatches shown with an asterisk. F5-20 was used as non-correcting control. n = 4.

Mentions: In the case of the F5-17 oligo, the EGFP+ cells seemed to achieve a proliferation rate similar to the uncorrected EGFP- cells eight days post-transfection, since the percentage of positive cells remained constant between 144 and 192 hrs (Fig. 4a) and when checked again after a few weeks of passaging (not shown). By doubling the amount of lipofectamine:oligo complex we were able to double the frequency of corrected cells to ~0.05% eight days post-transfection (Figure S4). We chose this time point to repeat our single-cell sorting assay and found that EGFP+ cells generated with the F5-17 oligo easily generated clonal populations. These clones were verified by sequencing and found to have the desired genome modification (Figure S5a). To verify that this modification was not due just to spontaneous mutations, we repeated the experiment with oligos that introduced two adjacent nucleotide changes, one of which would restore the ATG start codon of mEGFP (using either a T or FU base) and other of which would introduce either a second substitution or a single-base insertion (Fig. 5). As expected, efficiency of incorporation of double-mutant oligos was significantly lower than for single-substitution oligos, with the double-substitution being more efficient than the single-substitution-and-insertion. Interestingly, using the FU base had an adverse effect in the double-substitution, suggesting a more complex mismatch recognition effect when more than one substitution is involved. Although the correction efficiencies were low, it was possible to sort EGFP+ cells generated with all four oligos, and all clones proliferated normally and were verified by sequencing to be correctly modified(Figure S5b,c).


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)

Generating double mutants.Oligos complementary to the first start codon in the non-transcribed strand and containing a second mismatch, either an insertion (∧A) or substitution (-A), with silenced MSH2 and assayed 36 hrs after transfection. Targeting oligos shown in duplex 3′-’to 5′-, with PTO highlighted in gray and mismatches shown with an asterisk. F5-20 was used as non-correcting control. n = 4.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351460&req=5

pone-0036697-g005: Generating double mutants.Oligos complementary to the first start codon in the non-transcribed strand and containing a second mismatch, either an insertion (∧A) or substitution (-A), with silenced MSH2 and assayed 36 hrs after transfection. Targeting oligos shown in duplex 3′-’to 5′-, with PTO highlighted in gray and mismatches shown with an asterisk. F5-20 was used as non-correcting control. n = 4.
Mentions: In the case of the F5-17 oligo, the EGFP+ cells seemed to achieve a proliferation rate similar to the uncorrected EGFP- cells eight days post-transfection, since the percentage of positive cells remained constant between 144 and 192 hrs (Fig. 4a) and when checked again after a few weeks of passaging (not shown). By doubling the amount of lipofectamine:oligo complex we were able to double the frequency of corrected cells to ~0.05% eight days post-transfection (Figure S4). We chose this time point to repeat our single-cell sorting assay and found that EGFP+ cells generated with the F5-17 oligo easily generated clonal populations. These clones were verified by sequencing and found to have the desired genome modification (Figure S5a). To verify that this modification was not due just to spontaneous mutations, we repeated the experiment with oligos that introduced two adjacent nucleotide changes, one of which would restore the ATG start codon of mEGFP (using either a T or FU base) and other of which would introduce either a second substitution or a single-base insertion (Fig. 5). As expected, efficiency of incorporation of double-mutant oligos was significantly lower than for single-substitution oligos, with the double-substitution being more efficient than the single-substitution-and-insertion. Interestingly, using the FU base had an adverse effect in the double-substitution, suggesting a more complex mismatch recognition effect when more than one substitution is involved. Although the correction efficiencies were low, it was possible to sort EGFP+ cells generated with all four oligos, and all clones proliferated normally and were verified by sequencing to be correctly modified(Figure S5b,c).

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