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Results: Cas13a
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Protein cloning, expression and purification
We decided to compare 3 versions of Cas13a that were previously characterized in the literature1: Lbu, Lsh, and Lwa. We ordered the Lbu and Lsh plasmids from Addgene, and we cloned Lwa using Golden Gate assembly (sequence was taken from Gootenberg). Lbu and Lsh were expressed in E.coli Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in E.coli BL21 (DE3) star. We created three Biobricks from the Lwa sequence: BBa_K2323000 (containing the Lwa coding sequence and a Tphi terminator), BBa_K2323001 (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and BBa_K2323004 (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease BBa_K1319008 by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins.
We followed the purification protocols from literature, and found that although the His-purification and the tag cleavage steps worked as expected, the cation-exchange purification step failed, and we systematically lost our proteins. We still completed the size-exclusion purification, and our proteins with some amount of contamination. Protein purification took most of the first month of our project, due to the failure of the cation-exchange chromatography, but we eventually purified functional, if not perfectly clean, proteins.
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Proof-of-concept
To prove the functionality of Cas13a, we used the 16S rRNA sequence from E.coli as a targed sequence, since the 16S rRNA is highly conserved in all bacterial species, and can be easily extracted from bacterial cultures in large concentrations. For our first experiments, we used only 130 nucleotides of the 16s rRNA sequence and transcribed in vitro from a DNA template (since the whole 16s rRNA is 1500 nucleotides, therefore too large to be transcribed). Our crRNA DNA template was designed so that the target-binding region could easily be changed to detect new targets[scheme or link]. We found that both Lbu and Lwa were functional and degraded the read-out RNase Alert in presence of both the target and the crRNA. An example time plot is shown in Figure2, where the specific activity of Lbu was controlled by taking out the crRNA and Lbu, alternatively.
Lbu showed higher cleaving efficiency at equal concentrations compared to Lwa (this is in contrary to what was shown in Gootenberg et al.), and Lsh was not functional (we assume that the purification process inactivated the protein), see Figure 2. We therefore decided to use Lbu for the rest of our experiments.
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Figure 1: Plot of a typical experiment with 10nM Lbu Cas13a, 100nM crRNA, 50nM target, 185nM RNase Alert and 1U/µL RNase inhibitor. For analysis, we typically considered the fluorescence intensity of samples after 30 minutes, and normalized it to obtain the ratio of cleaved RNase Alert, assuming that our negative control (with neither crRNA and Cas13a) had 0% cleavage and our positive control (with RNaseA) had 100% cleavage. We should note that we occasionally found that high target concentrations led to above positive control signals (which could be due to degradation and lesser activity of RNaseA) and that low target concentrations led to below negative control signals (which could be due to noise at low fluorescence intensities).
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Figure 2: target RNA concentration was screened for all 3 Cas13a proteins and their matching crRNA. A conservative cut-off of at least 15% of the RNase Alert cleaved was chosen to determine the detection limit of our system.
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Figure 3: The activity of Lwa Cas13a was found to be similar before and after His purification.
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Interestingly, we found that Lwa was active even without purification: after lysing cells expressing the Cas13a, we used the supernatant in our detection system, and found similar activity as after purification, see Figure 3. This result, along with further characterization, showed us that Cas13a is a relatively robust enzyme that works in a variety of contexts.
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We screened the cleavage efficiency dependence on Cas13a and target concentrations, and found that for high Cas13a concentration, the background activity of Cas13a was overlaying with the target[plot of ratio vs Cas13a concentration] specific activation (Figure 4). As our device should detect low target RNA concentrations in less than 30minutes, we optimized the concentration of Cas13a: at high concentrations of the enzyme, the background activity hid the target-dependent signal; at low concentrations, the enzyme was too slow and a detectable signal could not be obtained in 30min unless large amounts of target RNA were added. A compromise was found at 10nM of Cas13a, and in these conditions, we found our target detection limit to be around 10nM (Figure 1).
Figure 2:At constant target RNA concentration, as Lbu Cas13a concentration increases, the background activity of the enzyme reduces the on/off ratio of activation by the target RNA.
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