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Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides.

Falgowski K, Falgowski C, York-Vickers C, Kmiec EB - Nucleic Acids Res. (2011)

Bottom Line: We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand.The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand.Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway.

View Article: PubMed Central - PubMed

Affiliation: Marshall Institute for Interdisciplinary Research, Marshall University, Robert C. Byrd Biotechnology Science Center, 1700 Third Avenue, Suite 220, Huntington, WV 25755, USA.

ABSTRACT
Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand. The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing.

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Average kanamycin-resistant colony counts for each recovery time point resulting from Kan49T-treated (A) and Kan49NT-treated (B) cells. (C) Average ampicillin-resistant colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution calculated from data from each recovery time point. (D) Average correction efficiencies calculated from data from all recovery time points for cells treated with no ODN, Kan49NT-PM, Kan49T-PM, Kan49NT and Kan49T. Correction efficiency is defined as the number of kanamycin-resistant colonies per 5 × 105 ampicillin-resistant colonies. ODN molarity was maintained at a 350:1 ODN to plasmid ratio for each experiment. For each reaction condition at least three independent experiments were conducted. **P < 0.001.
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Figure 2: Average kanamycin-resistant colony counts for each recovery time point resulting from Kan49T-treated (A) and Kan49NT-treated (B) cells. (C) Average ampicillin-resistant colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution calculated from data from each recovery time point. (D) Average correction efficiencies calculated from data from all recovery time points for cells treated with no ODN, Kan49NT-PM, Kan49T-PM, Kan49NT and Kan49T. Correction efficiency is defined as the number of kanamycin-resistant colonies per 5 × 105 ampicillin-resistant colonies. ODN molarity was maintained at a 350:1 ODN to plasmid ratio for each experiment. For each reaction condition at least three independent experiments were conducted. **P < 0.001.

Mentions: The gene editing reaction was begun by mixing the ODN with plasmid, pKSm4021, immediately prior to electroporation into E. coli strains DH10B. The cells were allowed to recover at 37 for 1 h, 2 h, 3 h or 4 h prior to plating on both ampicillin and kanamycin plates to observe possible changes to correction efficiency over time (see ‘Methods and Materials’ section). Antibiotic-resistant colonies were determined after 24 h of incubation at 37°C. The ODNs used in these experiments were 49 bases in length and annealed to either the T or replication lagging strand (Kan49T) or NT or replication leading (Kan49NT) strand of the mutant kanamycin resistance gene in plasmid pKSm4021 (Table 1). Average colony counts for each recovery time-point of cells treated with either Kan49T or Kan49NT are shown in Figure 2A and B. Cells treated with the Kan49T, as opposed to the Kan49NT, had about 100-fold more kanamycin-resistant colonies at each time-point. Figure 2C shows ampicillin colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution for each recovery point. Ampicillin colonies for both ODNs steadily increase over time and are within standard deviation of each other.Figure 2.


Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides.

Falgowski K, Falgowski C, York-Vickers C, Kmiec EB - Nucleic Acids Res. (2011)

Average kanamycin-resistant colony counts for each recovery time point resulting from Kan49T-treated (A) and Kan49NT-treated (B) cells. (C) Average ampicillin-resistant colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution calculated from data from each recovery time point. (D) Average correction efficiencies calculated from data from all recovery time points for cells treated with no ODN, Kan49NT-PM, Kan49T-PM, Kan49NT and Kan49T. Correction efficiency is defined as the number of kanamycin-resistant colonies per 5 × 105 ampicillin-resistant colonies. ODN molarity was maintained at a 350:1 ODN to plasmid ratio for each experiment. For each reaction condition at least three independent experiments were conducted. **P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113578&req=5

Figure 2: Average kanamycin-resistant colony counts for each recovery time point resulting from Kan49T-treated (A) and Kan49NT-treated (B) cells. (C) Average ampicillin-resistant colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution calculated from data from each recovery time point. (D) Average correction efficiencies calculated from data from all recovery time points for cells treated with no ODN, Kan49NT-PM, Kan49T-PM, Kan49NT and Kan49T. Correction efficiency is defined as the number of kanamycin-resistant colonies per 5 × 105 ampicillin-resistant colonies. ODN molarity was maintained at a 350:1 ODN to plasmid ratio for each experiment. For each reaction condition at least three independent experiments were conducted. **P < 0.001.
Mentions: The gene editing reaction was begun by mixing the ODN with plasmid, pKSm4021, immediately prior to electroporation into E. coli strains DH10B. The cells were allowed to recover at 37 for 1 h, 2 h, 3 h or 4 h prior to plating on both ampicillin and kanamycin plates to observe possible changes to correction efficiency over time (see ‘Methods and Materials’ section). Antibiotic-resistant colonies were determined after 24 h of incubation at 37°C. The ODNs used in these experiments were 49 bases in length and annealed to either the T or replication lagging strand (Kan49T) or NT or replication leading (Kan49NT) strand of the mutant kanamycin resistance gene in plasmid pKSm4021 (Table 1). Average colony counts for each recovery time-point of cells treated with either Kan49T or Kan49NT are shown in Figure 2A and B. Cells treated with the Kan49T, as opposed to the Kan49NT, had about 100-fold more kanamycin-resistant colonies at each time-point. Figure 2C shows ampicillin colony counts of Kan49T- and Kan49NT-treated cells at a 5 × 10−4 dilution for each recovery point. Ampicillin colonies for both ODNs steadily increase over time and are within standard deviation of each other.Figure 2.

Bottom Line: We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand.The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand.Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway.

View Article: PubMed Central - PubMed

Affiliation: Marshall Institute for Interdisciplinary Research, Marshall University, Robert C. Byrd Biotechnology Science Center, 1700 Third Avenue, Suite 220, Huntington, WV 25755, USA.

ABSTRACT
Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand. The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing.

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