Figure 2
From: Precise genome-wide base editing by the CRISPR Nickase system in yeast
Construction of the CRISPR Nickase system. (a) Scheme of the experiment. Cas9 nickase- and gRNA-expressing cassettes were integrated into a single plasmid that contained a donor DNA sequence. Transformants were cultivated in selective medium for 48 h from an OD600 of 10−5 (approximately 1.1 × 103 cells). (b) Genome editing efficiencies at the CAN1 gene. Efficiencies were calculated based on canavanine assays. The numbers indicate the distances (bp) of edited sites from the cleavage site. (c) Sequence analysis. Introduced stop codons (red) and unintended mutations (inverted) are represented. The numbers of observed sequences over the numbers of total sequenced strains are shown. All sequence results corresponding to Fig. 2b are described in Fig. S2. DS, downstream from the cleavage site. (d) Editing efficiencies at the different targeting sequence on the opposite strand of the CAN1 gene. Sequencing is shown in Fig. S3b. (e) Theoretical bases editable by the CRISPR Nickase system. The x-axis indicates the hypothetical editable distances from the nicking site. The y-axis shows theoretical editable bases (%) in the S. cerevisiae genome. The error bars show SEM based on at least three independent measurements. P values were determined by comparing between each sample and dead Cas9 control in each site, based on Tukey’s test. *P < 0.05, **P < 0.01.
