proof of concepts
In the presence of suvCas9s alone, GFP signal (indicating suvCas9) can only be observed in the nucleus,
indicating an enrichment of nuclear localization of suvCas9s. However, when small guide RNA
targeting the Actin message (sgActin) is co-expressed with suvCas9, GFP signal is relocated into
the cytoplasm. In contrast, after introducing PAMmers, which function as single-stranded DNA mimics,
the majority of suvCas9 proteins can now be stably sequestered in the cytoplasm.
Figure 1. Relocation into the cytoplasm of suvCas9 proteins can be orchestrated by single guide
RNAs targeting the Actin message (sgActin) and PAMmers.
As negative controls, when suvCas9 is expressed alone, or co-expressed with small guide RNA targeting
anti-sense message Actin mRNA sequence (sgNC), the GFP signal (indicating suvCas9) are strictly sequestered
in the nucleus, suggesting without the companion of sgRNA targeting sense Actin message, suvCas9 will be
translocated into the nucleus by its engineered NLS (nucleus localization sequence). As a positive control,
when yeasts are only transformed with a GFP expression construct, without the guidance of the NLS, the GFP
signal is limited within the cytoplasmic region.
Figure 2. Negative control and positive control for the relocation assay.