Fig. 3: Design and test of the strategy for reducing escape frequency by exploring CRISPR/Cas9 system to restrict occurrence of amber suppressor derived from tRNA^Tyr.

a Schematic diagram showing the design of CRISPR/Cas9-based “immunity” to the amber-suppressor tRNA derived from tRNA^Tyr. The escapee strain carries the C > G mutation on the non-template strand of the gene encoding tRNA^Tyr, which results in the production of tRNA^Tyr(CUA) with the G34 > C mutation in its anticodon. The 5’-AGAT-3’ sequence (G is labeled in yellow) in the genome of the escapee serves as the PAM site for SpCas9-NG when programmed with the designed gRNAs. In contrast, the wild-type strain has the corresponding 5’-ACAT-3’ sequence (C is labeled in blue-green) and lacks the PAM site. Thus, if an escapee arises within the population, it would be exposed to double-strand breaks enabled by the SpCas9-NG (labeled in light red) while the wild-type strain would be resistant to this event. Effect of CRISPR/Cas9-based “immunity” on escape frequency of OMeY-dependent auxotrophs based on CDC4_TAG325 (b) and CDC27_TAG520 (c) during the 12-day observation period. The expression of SpCas9-NG was induced by 2% (wt/vol) galactose. Strains that express the empty vector and the SpCas9-NG plasmid without the gRNAs serve as the controls. Escape frequencies were monitored from 3 to 12 days with error bars showing the mean ± SEM of six samples including three biological replicates that were conducted in duplicate. Source data are provided as a Source Data file.