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 dCas9(C)-TF-synVEGFR1 receptor's first chain and it is linked to 64, GAL4 and UAS Trans CMV enhancer.
Usage
This part is attached to NLS in first chain ofdCas9(C)-TF-synVEGFR1 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, two domains of dCas9 will be released and assembled forming active dCas9 which will be directed by Nanog gRNA and NLS with transcription activators to Nanog gene for boosting YAP-1 production.
this figure illustrates the structure of C- terminal domain of dCas9 in first chain of dCas9(C)-TF-synVEGFR1 receptor. .
Dry lab Characterization
The assembly of the dCas-9 domains are based on their affinity to each other. Also, we put in consideration the binding between the two domains in the presence of the guide RNA (gRNA). Indeed, we compared the two states by illustrating their interaction by the alpha fold 3 online tool. Then, we measured the binding stability between both domains using a prodigy Haddock software tool.
C-N dCas9 binding stability without gRNA
Alignment Plot
3D structure of dcas9 domains without gRNA
The alignment plot shows positive alignments between C\N dCas9 amino acid residues and the experimental structures. This indicates a valid dCas9 domains binding. The stability of binding was calculated using prodigy Haddock online tool. It gave us ΔG of -43.8 kcal mol-1 which is considered very high binding stability .
C-N dCas9 binding stability with gRNA
Alignment Plot
3D structure of dcas9 domains with gRNA
The alignment plot in gRNA presence is similar to that of dCas9 without gRNA which reflects that gRNA didn’t affect the domains binding state.
Characterization by Mathematical Modeling
The model provides the activation kinetics of the d-Cas9 system which occurs subsequent to the cleavage activity of TEV protease after its activation. The result shows increase in d-Cas9 activity which implies successful cleavage of the TEV protease for releasing the N and C terminal of the d-Cas9 system and its assembly based on parametric values from literature.
Graph (1). Illustrates the dimerization level (Blue line) that reaches steady state upon binding of VEGF to its receptor to activate TEV protease (Red line), The activation level of TEV protease reaches (14) to release d-Cas9 system .
Graph(2). Illustrates the released d-Cas9 system that activation reaches (240), upon activation of TEV protease .
Experimental Characterization
we digested dCas9(C)_NLS-Syn-VEGFR-1 grafetd with dCas9(C) and performed gel electrophoresis to detect the digested fragment.
This figure illustrates the digested dCas9(C)_NLS-Syn-VEGFR-1 prior to insert ligation .
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.
Paththamperuma C, Page RC. Fluorescence dequenching assay for the activity of TEV protease. Anal Biochem. 2022 Dec 15;659:114954. doi: 10.1016/j.ab.2022.114954. Epub 2022 Oct 18. PMID: 36265691; PMCID: PMC9662696.
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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