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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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(A) Average correction efficiencies for the entire time course of cells treated with Kan49NT, Kan49T, Kan49NT/3′InvdT and Kan49T/3′InvdT ODNs. 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. (B and C) Average number of kanamycin-resistant colonies resulting from each of the four RFLP categories was calculated from the RFLP percentages and the total number of kanamycin-resistant colonies of the time course (1 h, 2 h, 3 h and 4 h) from kanR49NT, kanR49T, kanR49NT/3′InvdT and kanR49T/3′ODN-treated resistant colonies. Average RFLP percentages were calculated from 10–20 plasmid isolates resulting from each ODN-treated kanamycin-resistant colony populations. Three independent experiments were performed to determine average number of kanamycin-resistant colonies for each recovery time point. **P < 0.001.
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Figure 5: (A) Average correction efficiencies for the entire time course of cells treated with Kan49NT, Kan49T, Kan49NT/3′InvdT and Kan49T/3′InvdT ODNs. 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. (B and C) Average number of kanamycin-resistant colonies resulting from each of the four RFLP categories was calculated from the RFLP percentages and the total number of kanamycin-resistant colonies of the time course (1 h, 2 h, 3 h and 4 h) from kanR49NT, kanR49T, kanR49NT/3′InvdT and kanR49T/3′ODN-treated resistant colonies. Average RFLP percentages were calculated from 10–20 plasmid isolates resulting from each ODN-treated kanamycin-resistant colony populations. Three independent experiments were performed to determine average number of kanamycin-resistant colonies for each recovery time point. **P < 0.001.

Mentions: Cells were transfected with ODNs (Kan49NT/3′InvdT and Kan49T/3′InvdT) identical to the Kan49NT and Kan49T ODNs with the exception of a 3′-end inverted nucleotide, then allowed to recover for 1 h, 2 h, 3 h or 4 h prior to plating for antibiotic selection. Correction efficiencies (number of kanamycin resistant colonies per 5 × 105 ampicillin-resistant colonies) were calculated from triplicate experiments for Kan49NT/3′InvdT and Kan49T/3′InvdT-treated cells to be 0.35 ± 0.30 and 6.2 ± 4.5, respectively, and once again presented as an average (Figures 4C and 5A). When the Kan49NT/3′InvdT correction efficiency was compared to the Kan49NT correction efficiency of 0.52 ± 0.06 (P = 0.15), there was only a modest change based mostly on the low numbers achieved using the NT ODN. Alternatively, the blocked 3′-end of the Kan49T/3′InvdT caused almost an 8-fold reduction in the correction efficiency when compared the Kan49T ODN correction efficiency of 50.1 ± 6.7 (P = 0.0002).Figure 5.


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)

(A) Average correction efficiencies for the entire time course of cells treated with Kan49NT, Kan49T, Kan49NT/3′InvdT and Kan49T/3′InvdT ODNs. 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. (B and C) Average number of kanamycin-resistant colonies resulting from each of the four RFLP categories was calculated from the RFLP percentages and the total number of kanamycin-resistant colonies of the time course (1 h, 2 h, 3 h and 4 h) from kanR49NT, kanR49T, kanR49NT/3′InvdT and kanR49T/3′ODN-treated resistant colonies. Average RFLP percentages were calculated from 10–20 plasmid isolates resulting from each ODN-treated kanamycin-resistant colony populations. Three independent experiments were performed to determine average number of kanamycin-resistant colonies for each recovery time point. **P < 0.001.
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Figure 5: (A) Average correction efficiencies for the entire time course of cells treated with Kan49NT, Kan49T, Kan49NT/3′InvdT and Kan49T/3′InvdT ODNs. 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. (B and C) Average number of kanamycin-resistant colonies resulting from each of the four RFLP categories was calculated from the RFLP percentages and the total number of kanamycin-resistant colonies of the time course (1 h, 2 h, 3 h and 4 h) from kanR49NT, kanR49T, kanR49NT/3′InvdT and kanR49T/3′ODN-treated resistant colonies. Average RFLP percentages were calculated from 10–20 plasmid isolates resulting from each ODN-treated kanamycin-resistant colony populations. Three independent experiments were performed to determine average number of kanamycin-resistant colonies for each recovery time point. **P < 0.001.
Mentions: Cells were transfected with ODNs (Kan49NT/3′InvdT and Kan49T/3′InvdT) identical to the Kan49NT and Kan49T ODNs with the exception of a 3′-end inverted nucleotide, then allowed to recover for 1 h, 2 h, 3 h or 4 h prior to plating for antibiotic selection. Correction efficiencies (number of kanamycin resistant colonies per 5 × 105 ampicillin-resistant colonies) were calculated from triplicate experiments for Kan49NT/3′InvdT and Kan49T/3′InvdT-treated cells to be 0.35 ± 0.30 and 6.2 ± 4.5, respectively, and once again presented as an average (Figures 4C and 5A). When the Kan49NT/3′InvdT correction efficiency was compared to the Kan49NT correction efficiency of 0.52 ± 0.06 (P = 0.15), there was only a modest change based mostly on the low numbers achieved using the NT ODN. Alternatively, the blocked 3′-end of the Kan49T/3′InvdT caused almost an 8-fold reduction in the correction efficiency when compared the Kan49T ODN correction efficiency of 50.1 ± 6.7 (P = 0.0002).Figure 5.

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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