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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) Representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301 bp band. The conversion of the target base will result in the loss of a cut site in the 1301 bp band thereby producing a 1534 band. (B) RFLP patterns of the 20 kanamycin-resistant plasmid population isolates resulting from Kan49T- or Kan49NT-treated cells for the 1 h recovery time point (kanR49T #1-20 and kanR49NT #1-20). Isolate kanS is the RFLP from a mutant-plasmid population isolated from an ampR/kanS colony from the respective Kan49T- and Kan49NT-treated cells. Four different RFLP patterns are shown, A–D. (C) Representative data of the NT and T strand sequences obtained from each of the RFLP patterns. The wild-type base pairing at the target site is T–A, the mutant base pairing is G–C, while the converted base pairing is C–G.
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Figure 3: (A) Representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301 bp band. The conversion of the target base will result in the loss of a cut site in the 1301 bp band thereby producing a 1534 band. (B) RFLP patterns of the 20 kanamycin-resistant plasmid population isolates resulting from Kan49T- or Kan49NT-treated cells for the 1 h recovery time point (kanR49T #1-20 and kanR49NT #1-20). Isolate kanS is the RFLP from a mutant-plasmid population isolated from an ampR/kanS colony from the respective Kan49T- and Kan49NT-treated cells. Four different RFLP patterns are shown, A–D. (C) Representative data of the NT and T strand sequences obtained from each of the RFLP patterns. The wild-type base pairing at the target site is T–A, the mutant base pairing is G–C, while the converted base pairing is C–G.

Mentions: Twenty random kanamycin-resistant colonies were picked from the kanamycin plates and grown at 37°C for 8 h in LB broth under kanamycin selection. Plasmid populations were isolated from each individual culture and then digested with BfaI. In Figure 3A, a representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301-bp band. The conversion of the target base will result in the loss of a cut site in the 1301-bp band, thereby producing a 1534 band.Figure 3.


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) Representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301 bp band. The conversion of the target base will result in the loss of a cut site in the 1301 bp band thereby producing a 1534 band. (B) RFLP patterns of the 20 kanamycin-resistant plasmid population isolates resulting from Kan49T- or Kan49NT-treated cells for the 1 h recovery time point (kanR49T #1-20 and kanR49NT #1-20). Isolate kanS is the RFLP from a mutant-plasmid population isolated from an ampR/kanS colony from the respective Kan49T- and Kan49NT-treated cells. Four different RFLP patterns are shown, A–D. (C) Representative data of the NT and T strand sequences obtained from each of the RFLP patterns. The wild-type base pairing at the target site is T–A, the mutant base pairing is G–C, while the converted base pairing is C–G.
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Figure 3: (A) Representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301 bp band. The conversion of the target base will result in the loss of a cut site in the 1301 bp band thereby producing a 1534 band. (B) RFLP patterns of the 20 kanamycin-resistant plasmid population isolates resulting from Kan49T- or Kan49NT-treated cells for the 1 h recovery time point (kanR49T #1-20 and kanR49NT #1-20). Isolate kanS is the RFLP from a mutant-plasmid population isolated from an ampR/kanS colony from the respective Kan49T- and Kan49NT-treated cells. Four different RFLP patterns are shown, A–D. (C) Representative data of the NT and T strand sequences obtained from each of the RFLP patterns. The wild-type base pairing at the target site is T–A, the mutant base pairing is G–C, while the converted base pairing is C–G.
Mentions: Twenty random kanamycin-resistant colonies were picked from the kanamycin plates and grown at 37°C for 8 h in LB broth under kanamycin selection. Plasmid populations were isolated from each individual culture and then digested with BfaI. In Figure 3A, a representative map of expected DNA bands resulting from a restriction digest of a mutant plasmid, converted plasmid and mixed plasmid population is presented. The BfaI enzyme recognizes the mutant base present at the target site, resulting in the appearance of a 1301-bp band. The conversion of the target base will result in the loss of a cut site in the 1301-bp band, thereby producing a 1534 band.Figure 3.

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.

Show MeSH