Part:BBa_K1923011
Gal4BD-NLS-FLAG-EGFP-dCas9-NLS-Gal4AD encoding gene
Section below is the contribution Team Tsinghua made to previous parts (https://parts.igem.org/Part:BBa_K1323002, https://parts.igem.org/Part:BBa_K1493504 and https://parts.igem.org/Part:BBa_K1470002). We characterized these parts by transforming the part into E. coli and validated its sequence using enzymatic digestion as well as sequencing, as is supported by Figure 1. We functionally improved these parts by fusing three parts together (dCas9 from BBa_K1323002, GFP from BBa_K1493504, and Gal4BD from BBa_K1470002). As is indicated below in Figure 2, the nuclear localization of dCas9 protein can be well visualized. As for the shipment information, we submitted this part in the form of BBa_K1923007, the shipment information of which can be seen here https://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_K1923007. We do not intend to use the part BBa_K1923007 as part of the silver medal criteria now.
Figure 1. Enzymatic digestion of three parts selected to be improved.
This composite part is a fusion protein composed of GAL4, triple FLAG tag, two SV40 nuclear localization sequence,dCas9 and GFP. Triple FLAG tag and GFP is designed for observation and expression detection. The protein produced by this parts can be sequestered in the cytoplasm with the help of sgRNA and PAMmer, utilizing dCas9's mRNA binding ability. However, when there is a mutation in the target sequence of the mRNA, sv40 NLS would drag this protein into the nuclear. Then GAL4 would active the gene downstream UAS promoter. Thus this parts can be used for gene mutation surveillance [1].
Figure 2. 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 3. 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2394
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4673
Illegal XhoI site found at 220
Illegal XhoI site found at 567 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 139
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