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. 2023 Mar 20;51(7):3307–3326. doi: 10.1093/nar/gkad186

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Stalled replication rescue at less stable roadblocks. (A) Schematic representation of dCas9-gRNA mismatch complexes binding to 83-mer dsDNA oligonucleotides in single-molecule lifetime assays. (B) Lifetime distributions of dCas9-MMgRNA complexes binding to 83-mer dsDNA. Mean lifetime for each complex: dCas9-gRNA80, 8.6 ± 0.1 min (S.E.M.; n= 1414 events); dCas9-gRNA60, 8.3 ± 0.1 min (n= 619 events); dCas9-gRNA40, 1.0 ± 0.1 min (n= 249 events); dCas9-gRNA20, 1.0 ± 0.1 min (n= 103 events). Comparison of distributions was conducted by Kruskal-Wallis test for multiple comparisons with Dunn's procedure, where ** and **** denote statistical significance with p ≤ 0.01 and 0.0001, respectively. Absence of markers denotes no significance difference (p > 0.05). (C) The efficiency of replication distributions of DNA templates following pre-incubation with dCas9-gRNA complexes. Mean efficiencies for each complex: dCas9-gRNA100, 0.02 ± 0.01%; dCas9-gRNA80, 0.5 ± 0.1%; dCas9-gRNA60, 0.6 ± 0.1%; dCas9-gRNA40, 2.0 ± 0.3%; dCas9-gRNA20, 3.5 ± 0.3%; No dCas9 complexes, 4.0 ± 0.3%. Comparison of the mean replication efficiency was done using one-way ANOVA with Tukey's comparison post-hoc test, where ** and *** denote statistical significance with p ≤ 0.01 and 0.001, respectively. Absence of markers indicates no significance difference (p > 0.05). (D) Pause duration distributions in the presence of dCas9-MMgRNA complexes and Rep-AF647. Mean pause durations were determined from fitting a single-exponential decay function to the data of each complex: dCas9-gRNA80, (40 ± 20 s (S.E.M.), n = 51 pauses, replication efficiency of 3 ± 1%); dCas9-gRNA60, (20 ± 6 s, n = 72, 3 ± 1%); dCas9-gRNA40, (30 ± 14 s, n = 16, 4 ± 1%). dCas9-gRNA100 data are duplicated from Figure 5A (dCas9-cgRNA1 and 10 nM Rep-AF647). Pause durations represent pauses where Rep-AF647 intensity was observed above the threshold. Comparison of distributions was done by Kruskal-Wallis test for multiple comparisons with Dunn's procedure, where ** denotes statistical significance with p ≤ 0.01 and ns denotes no significance difference (p > 0.05).