Difference between revisions of "Part:BBa K1923008"

Latest revision as of 21:30, 19 October 2016

sgRNA targeting actin sense message (sgActin)

This part expresses small guide RNA targeting yeast Actin mRNA. With the assistance of PAMmer, a nucleotide oligo containing NGG sequence, it is able to help dCas9 to bind to the Actin mRNA transcripts, thus sequesters dCas9 in the cytoplasm. The transcription of this sgRNA is driven by tRNATyr promoter. HDV ribosome is a self-splicing element, which would process sgRNA to a mature transcript ^[1].

For more information, please refer to the Wiki page of Team Tsinghua.

Figure 1. Relocalization into the cytoplasm of suvCas9 proteins can be orchestrated by single guide RNAs targeting the Actin message (sgActin) and PAMmers.

In order to test that this construct is indeed functional, we performed a nuclear relocalization assay where we essentially compared the difference in the distribution of dCas9 with fused GFP in the yeasts. We transformed either dCas9 alone or dCas9 with sgActin expressing plasmid or dCas9 with both sgActin expressing plasmid as well as PAMmer into the yeast, and observed GFP signal under a fluorescence microscopy. As is indicated in Figure 1, relocalization into the cytoplasm of suvCas9 proteins can be orchestrated by single guide RNAs targeting the Actin message (sgActin) and PAMmers. Specifically, 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 2. Negative control and positive control for the relocation assay.

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.

Reference:
[1] Nelles D A, Fang M Y, O’Connell M R, et al. Programmable RNA tracking in live cells with CRISPR/Cas9[J]. Cell, 2016, 165(2): 488-496.

Sequence and Features