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Fig. 4 | Genome Biology

Fig. 4

From: Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

Fig. 4

Mismatched or truncated sgRNAs reduce target residence of Cas9-sgRNA in vitro at the gHRC2, gHRC4, gEJC5, and gEJW7 sites. The schematics for the target binding, cleavage, and dissociation reaction were shown on the top. a DNA cleavage by SpCas9-20-nt perfectly matched sgRNAs and SpCas9-sgRNA variants at different time points of the reaction as indicated. Fluorescence-labeled DNA targets were 620 bp or 623 bp as shown. DNA cleavage from the reaction was detected on 2% denaturing agarose gel. b Residence of SpCas9-sgRNAs including SpCas9-20-nt control and its variants on cleaved DNA. SpCas9-sgRNAs were incubated with fluorescence-labeled target DNAs from a for 24 h. DNA bound with SpCas9-sgRNAs and unbound DNA were resolved by 4–20% native PAGE gel. c Target residence of dSpCas9-sgRNAs including dSpCas9-20nt control and its variants. dSpCas9-sgRNAs were incubated with fluorescence-labeled target DNAs from a for 1 and 24 h. DNA bound with dSpCas9-sgRNAs and unbound DNA were resolved by 4–20% native PAGE gel. d Cleavage of DNA released from the dSpCas9-sgRNA-DNA ternary complex by competing SpCas9-20nt sgRNA. The preassembled dSpCas9-20-nt sgRNA and dSpCas9-sgRNA variant complexes were incubated with fluorescence-labeled target DNAs from a for 1 h. The preassembled SpCas9-20nt perfectly matched sgRNA complex was added to compete for binding to DNA targets released from the dSpCas9-sgRNA-DNA complex and cleave DNA for 6 and 24 h. DNA cleavage from the reaction was detected on 2% denaturing agarose gel. The efficiency of target cleavage and target dissociation was calculated as the intensity ratio of cut DNA to total DNA in a and d and the intensity ratio of unbound DNA to total DNA in b and c, respectively. The values of these ratios were shown in percentages under each DNA gel

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