Part:BBa_K4818026

dCas9

dCas9, or "dead Cas9," is a modified version of the Cas9 protein used in the CRISPR-Cas9 genome editing system. The "dead" in dCas9 refers to the fact that this variant of the Cas9 protein lacks its usual nuclease activity, which is responsible for cutting DNA. Instead, dCas9 retains its ability to bind to specific DNA sequences but cannot cleave or edit the DNA.

Here are the key features and uses of dCas9:

1. **Loss of Nuclease Activity**: In the original Cas9 protein, the nuclease activity is essential for creating double-strand breaks in the target DNA, which can be used for gene editing. In dCas9, specific amino acid changes or mutations are introduced to inactivate this cutting function. This means that dCas9 can still bind to DNA but cannot introduce DNA breaks.

2. **Gene Regulation**: dCas9 is primarily used as a tool for gene regulation rather than genome editing. By attaching various regulatory elements to dCas9, researchers can direct it to specific genes or DNA sequences in the genome and modulate gene expression without changing the underlying DNA sequence. This is often referred to as "CRISPR interference" or "CRISPRi."

3. **Applications**: dCas9 has a wide range of applications in molecular biology and biotechnology. It can be used to study gene function, investigate gene networks, and control gene expression. Researchers can attach repressor or activator domains to dCas9 to inhibit or enhance gene transcription, respectively, at specific target genes. This allows for precise control over which genes are turned on or off in a cell.

4. **Epigenome Editing**: dCas9 can also be used for epigenome editing, which involves modifying the epigenetic marks on DNA or histones without altering the DNA sequence itself. This can influence gene expression by changing the accessibility of genes to the transcriptional machinery.

5. **RNA Targeting**: In addition to DNA binding, dCas9 can also be directed to target and manipulate RNA molecules. This is known as "CRISPR interference of RNA" (CRISPRi-RNA). dCas9 can be guided to specific RNA sequences to interfere with RNA stability or translation, offering a way to control gene expression post-transcriptionally.

In summary, dCas9 is a modified version of the Cas9 protein that lacks nuclease activity, making it a valuable tool for precise gene regulation, epigenome editing, and RNA targeting. It has revolutionized the field of molecular biology by enabling researchers to control gene expression and study gene function with a high degree of specificity and accuracy.

Improvement by AFCM-Egypt 2024

dCas9 is a modified version of Cas9 enzyme, originally designed to cut DNA. dCas9 has been changed in a way that makes it incapable of performing this process of cutting DNA. As a result, it is pretty useful in regulating gene expression. Cas9 is prone to produce unwanted DNA cutting, while dCas9 can be utilized to induce or suppress the activity of any certain genes without disturbing the coding sequence.

However, one of the biggest challenges was the regulation of dCas9 activity and to serve this purpose, we integrated CRISPR-dCas9 technology into a novel synthetic receptor based-system called dCas9-TF Syn-VEGFR-1\2. So that we could improve ( BBa_K4818026) which was designed by iGEM23_INSAENSLyon1 .

As a result, dCas9 activity will be linked and regulated through the receptor state in response to the VEGF concentration within the microenvironment of our engineered cells expressing the system.

Furthermore, to limit the basal activity of the CRISPR-dCas9 that is usually associated with gene off targeting effects. According to that, we optimized the safety of dCas9 based gene regulation strategies through splitting dCas9 protein into two non-functional domains including: one part containing the C-terminal domain (c) dCas9-TF and another part, the N-terminal domain (N) dCas9. Each domain is attached to a different chain of the receptor forming dCas9 (C/N)-TF Syn-VEGFR-1\2 system. VEGF dependent dimerization of the two chains of the receptor will mediate release of the two non-functional domains into the cytoplasm and spontaneous assembly of the two fragments into an effector complex.

Moreover, the translocation of the dCas9-TF to target genes is mediated through nuclear translocation signal (NLS) and guide RNA which constantly guides dCas9 coupled with other transcription activators to target genes. In our design dCas9-TF activity will be recruited to Nanog enhancer gene locus which lies within chromosome 11 upstream to YAP-1 gene, regulating cell differentiation and proliferation.

This figure illustrates VEGF dependent dimerization of the two domains of dCas9 .

Dry lab Characterization by AFCM-Egypt2024

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 by AFCM-Egypt2024

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 .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3378
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]