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.
We also developed a curve that modeled unbounded inhA DNA over time, showing that as time goes on, the dCas9 is able to inhibit the inhA DNA, and as the dCas9 reaches a certain saturation point, the graph goes towards an asymptote at 0.
The initial concentration of inhA DNA undergoes a gradual decrease as it becomes bound by the dCas9-sgRNA complex during the CRISPRi reaction. Initially, the unbound inhA DNA is available for transcription, leading to GFP expression. However, as the reaction progresses, dCas9 binds to the target sequences on the inhA DNA, forming a dCas9-sgRNA-GFP complex. This binding reduces the amount of unbound inhA DNA, inhibiting further transcription. The rate at which the inhA DNA becomes bound follows a positive logarithmic pattern, reflecting the progressive, saturating effect of dCas9 inhibition over time.
We recreated the graph of RFU expression of our wetlab’s experimental M. tb CRISPRi results. As the graph progresses, you would see that at lower percentages of bound inhA DNA, the RFU values remain high, reflecting active GFP expression. However, as the binding percentage rises, especially after the initial stages of the reaction, there will be a noticeable plateaus in RFU, correlating with the inhibition of GFP transcription. To evaluate the effectiveness of our wetlab experimentation (see CRISPRi), we utilized a line-of-best-fit between GFP expression and fluorescence to correlate the reactions, represented by the equation RFU = 6.1[GFP] for Lambert High School’s plate reader and RFU = 231.1 [GFP] for the plate reader used at the Georgia Institute of Technology. By converting our CRISPRi DNA concentration to fluorescence, the similarity presented by the logarithmic shape of our model and our experimental results prove the accuracy of our wetlab. The minor discrepancies in the scales of the graphs could be attributed to potential pipetting errors or differences in the sensitivity or calibration of the plate readers we used.
This graph effectively illustrates the relationship between the percentage of bound inhA DNA and both the experimental and theoretical modeled RFU values. The observed stagnation and decline in fluorescence units with the increase in bound DNA percentage demonstrates the successful application of the CRISPRi mechanism – as the shapes are relatively similar to the wetlab results – validating the model's predictions and emphasizing the efficiency of dCas9 in gene regulation.
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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