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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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Chemically-modified base analogs.(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.
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pone-0036697-g002: Chemically-modified base analogs.(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.

Mentions: Previous work has shown that the MMR machinery plays a significant role recognizing and removing the mutation caused by the oligo incorporation event. Currently, the only way around this is by either completely knocking down one of the main MMR proteins (e.g. MSH2) or by transient silencing with RNAi [29]. Our group recently showed an alternative strategy in E. coli, where the oligo contains chemically modified bases that avoid mismatch repair recognition. We tested oligos complementary to the non-transcribed strands of the two potential start codons of mEGFP while varying the mismatched base (Fig. 1a, Fig. 2). The best replacement for the T-T mismatch on the first start codon was 2′-Fluorouracil (FU), while for the A-A mismatch on the second start codon 2-Aminopurine (AM) was best, each giving a ~2-fold increase in mEGFP correction efficiency (Fig. 2a,b). To test whether the increased efficiency in mEGFP correction by the modified bases was due to avoidance of MMR, we transfected cells with validated shRNAs plasmids targeting MSH2 and MLH1(Table S2), then tested the mEGFP correction efficiency using oligos with the different modified bases. Downregulating either MSH2 or MLH1,as confirmed by western blotting (Figure S3a), lead to a ~2-fold increase in mEGFP targeting efficiency in both the T-T and the A-A mismatched oligos (Fig. 2c,d). However, this increase was significantly lower for FU-T (Student t-test p-val = 1.26E-5 for MLH1, 3.42E-5 for MSH2) and somewhat lower for AM-A mismatch (p-val = 0.07 for MLH1). Interestingly, FA seems to be more strongly recognized by MMR than the natural base, suggesting an alternate mechanism is producing the increase in targeting efficiency observed. These results were verified by targeting MSH2 and MLH1 with siRNAs (Figure S3b,c). Thus, silencing MSH2 and MLH1 decreased the gap in targeting frequencies seen between the natural and modified bases, suggesting that the increase in targeting efficiency by oligos containing modified bases can be explained in part due to avoidance of MMR.


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)

Chemically-modified base analogs.(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351460&req=5

pone-0036697-g002: Chemically-modified base analogs.(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.
Mentions: Previous work has shown that the MMR machinery plays a significant role recognizing and removing the mutation caused by the oligo incorporation event. Currently, the only way around this is by either completely knocking down one of the main MMR proteins (e.g. MSH2) or by transient silencing with RNAi [29]. Our group recently showed an alternative strategy in E. coli, where the oligo contains chemically modified bases that avoid mismatch repair recognition. We tested oligos complementary to the non-transcribed strands of the two potential start codons of mEGFP while varying the mismatched base (Fig. 1a, Fig. 2). The best replacement for the T-T mismatch on the first start codon was 2′-Fluorouracil (FU), while for the A-A mismatch on the second start codon 2-Aminopurine (AM) was best, each giving a ~2-fold increase in mEGFP correction efficiency (Fig. 2a,b). To test whether the increased efficiency in mEGFP correction by the modified bases was due to avoidance of MMR, we transfected cells with validated shRNAs plasmids targeting MSH2 and MLH1(Table S2), then tested the mEGFP correction efficiency using oligos with the different modified bases. Downregulating either MSH2 or MLH1,as confirmed by western blotting (Figure S3a), lead to a ~2-fold increase in mEGFP targeting efficiency in both the T-T and the A-A mismatched oligos (Fig. 2c,d). However, this increase was significantly lower for FU-T (Student t-test p-val = 1.26E-5 for MLH1, 3.42E-5 for MSH2) and somewhat lower for AM-A mismatch (p-val = 0.07 for MLH1). Interestingly, FA seems to be more strongly recognized by MMR than the natural base, suggesting an alternate mechanism is producing the increase in targeting efficiency observed. These results were verified by targeting MSH2 and MLH1 with siRNAs (Figure S3b,c). Thus, silencing MSH2 and MLH1 decreased the gap in targeting frequencies seen between the natural and modified bases, suggesting that the increase in targeting efficiency by oligos containing modified bases can be explained in part due to avoidance of MMR.

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