Part:BBa_K5036004
d-CAS9(C)
Part Description
It is a modified version of the CRISPR-Cas9 gene editing tool which cannot cut DNA. Instead, it can bind to a specific DNA sequence guided by an RNA molecule so it can be fused to transcriptional activators or repressors. In our model dcas9 was divided into N-terminal and C-terminal fragments. our dCas9 (c) is attached to our sCas9 synRTK receptor's first chain and it is linked to NLS,VP64,GAL4 and UAS Trans CMV enhancer.
Usage
This part is attached to NLS in first chain of dCas9-synRTK receptor and won’t be released until cleavage of TCS(Q,G) and TCS(Q,L) which prevents the self-assembly of the two domains of dCas9 therefore providing control over the transcription activity. Once the receptor is activated, it'll be directed by Nanog gRNA to join with transcription activators, boosting YAP production.
this figure illustrates the structure of C- terminal domain of dCas9 in first chain of dCas9-synRTK receptor. .
literature characterization
In this study,The researchers used four strains of E. coli bacteria: DH5alpha and JM109 (both dam+/dcm+), and JM110 and GM2163 (dam-/dcm-). They introduced two plasmids, pRA-cas9 and pRA-dcas9, into each strain. These plasmids express Cas9 or dCas9 proteins under the control of a powerful, constantly active promoter called PgroESL.
(Fig.a)the presence or absence of DNA methylation (dam+/dcm+ vs. dam-/dcm-) didn't significantly affect the toxicity of Cas9 or dCas9 proteins. However, there was a slightly lower success rate in transforming dam-/dcm- strains with plasmids containing cas9/dcas9 genes compared to strains with normal methylation. This suggests that DNA methylation might play a minor role in the efficiency of plasmid transformation with these specific genes
In order to determine expression of Cas9/dCas9 in each strain of E. coli, the researcher separated the cell extracts using electrophoresis on a gel (SDS-PAGE). Finally, they visualized the proteins using two methods: staining the gel with Coomassie blue and Western blotting.
(Fig.b&c)The experiment confirmed the production of Cas9 and dCas9 proteins in all E. coli strains containing the relevant plasmids. These proteins appeared as a band around 160 kDa on the gel and were absent in strains with empty vector controls. Interestingly, the levels of Cas9/dCas9 expression were slightly higher in strains with normal DNA methylation (dam+/dcm+) compared to those lacking methylation (dam-/dcm-). Additionally, expression levels varied between strains, with DH5alpha showing the highest and GM2163 showing the lowest. Despite lower protein expression in dam-/dcm- strains, the results suggest a possibility of slightly greater toxicity associated with Cas9/dCas9 in these strains compared to dam+/dcm+ strains.
Reference
Misra, C. S., Bindal, G., Sodani, M., Wadhawan, S., Kulkarni, S., Gautam, S., ... & Rath, D. (2019). Determination of Cas9/dCas9 associated toxicity in microbes. BioRxiv, 848135.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 577
Illegal NgoMIV site found at 650
Illegal NgoMIV site found at 1135
Illegal NgoMIV site found at 2044 - 1000COMPATIBLE WITH RFC[1000]
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