Part:BBa_K5096071
sgRNA 70 inhA
The sequence encodes the complete sgRNA70, which guides the dCas9 protein to a specific genomic site. In this case, sgRNA70 targets the inhA region of the M. tuberculosis genome. The binding sequence of sgRNA70 is derived from the inhA gene, chosen for its low mutation rate and essential role in the pathogenicity and survival of M. tuberculosis. The inhA gene encodes NADH-dependent enoyl-acyl carrier protein reductase, a crucial enzyme in mycolic acid synthesis, a vital component of the bacterial cell envelope of M. tuberculosis (Marrakchi et al., 2014). By inhibiting mycolic acid synthesis through the CRISPRi system, sgRNA70 seeks to destabilize the bacterial cell wall, thereby weakening the pathogen.
The CRISPR sgRNA design tool from Benchling was utilized to generate the guide sequence, along with the sgRNA-tracr and sgRNA repeat from Marshall et al. (2018).
To validate the efficacy of sgRNA70, repression of the target inhA construct was tested in a TXTL cell-free system. After calculating the percent repression, sgRNA70 demonstrated a 60.4% reduction in fluorescence compared to the positive control. This significant decrease indicates that sgRNA70 effectively downregulates the inhA gene, potentially disrupting the pathogenicity of M. tuberculosis and improving efforts to combat the bacteria.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 36
Illegal NheI site found at 59 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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