Results

In vivo experiment of dCas9 localization guided by sgRNA in S. cerevisiae

In order to achieve the surveillance function of the whole circuitry, it is of great significance to control
the precise spatial distribution of suvCas9. With suvCas9 fused with GFP, we can easily detect the cellular
localization of suvCas9 by fluorescent microscopy. We assembled suvCas9 and beta-Actin transcript targeted
sgRNA into yeast protein and gRNA expression plasmid respectively. PAMmer is directly synthesized from IDT.
Considering the unique characteristics of yeast, suvCas9 and sgRNA expression plasmid are transformed by the
lithium acetate/PEG-4000 method, while PAMmer by electroporation. We observed perfect nucleus localization
of suvCas9 without the presence of PAMmer, indicating the NLS on suvCas9 as well as the DNA binding domain
fused to dCas9 are powerful enough to sustain suvCas9 in the nucleus. After the co-transformation of sgRNA
expression plasmid and PAMmer, a certain population of yeast cells showed obvious appearance of cytoplasmic
suvCas9 complex. The control group with sgRNA targeted to non-sense strand did not show suvCas9 complex
transfer from nucleus to cytoplasm, which implied that the specificity of suvCas9 is double-determined by
sgRNA as well as PAMmer. More importantly, suvCas9 protein, sgRNA and PAMmer form a complex that can be
trapped in the cytoplasm by sgRNA-targeted mRNA recognition and binding ability. Another important
parameter we are currently calculating is the percentage of cells that exhibit nucleus to cytoplasm
transfer, which is an essential quantity required for our modeling process. However, comparing the
percentage of GFP-expressing population before and after electroporation, a significant drop was
witnessed, suggesting the optimization of PAMmer transformation and method and the development of
ssDNA-generation system.

In vitro evidence for the mRNA binding ability of suvCas9 complex

Aside from in vivo experiment, we intended to prove the mRNA binding ability of suvCas9 complex
by gel shift. We conducted the in-vitro transcription of sgRNA by T7 promoter. And we are now working
on the expression and purification of suvCas9 protein. Due to its relatively large size and complexity,
we decided to express suvCas9 protein in yeast. After purifying the protein complex, we would label
in-vitro transcribed mRNA with radioactive nucleotides and perform gel shift experiment on RNA gel.
The expected result is that the presence of PAMmer, sgRNA and suvCas9 protein change the mobility of
mRNA and cause visible gel shift.

Cellular suicidal system in S. cerevisiae

To test if the GAL4 binding domain and its activation domain fused dCas9 can actually activate downstream
gene expression, we initially tested the background yeast one-hybrid strain with suvCas9. The background
yeast strain we used contains SPAL:URA3 engineered into S. cerevisiae genome. Interaction between suvCas9
protein complex and SPAL promoter will drive the expression of URA3, therefore ensure the viability of
yeast on autotrophy selection plates, SC-URA. The results indicated that successful expression of as well
as the nucleus localization of suvCas9 protein complex, would guarantee downstream gene activation based
on yeast one-hybrid system. This is the premise of suicidal gene activation once the potential mutation
occurs and suvCas9 recover its nuclear targeting capability.

proof of concepts

suvCas9 relocalization

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.