Part:BBa_K3602007:Experience

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Applications of BBa_K3602007

rs6983267 G target activates Cas13a

It is seen that Cas13a is dependent on the sgRNA and thus that the sgRNA work as intended. In Figure 3, the sgRNA and Cas13a (BBa_K3602020) are co-incubated. Then the target (BBa_K3602007) is added to the samples along with ribosomal RNA (rRNA). The samples are then incubated for 15, 40 mins or 2 hours to activate collateral cleavage of Cas13a. The activation of Cas13a is seen as a smear indicating that the Cas13a is activated and starting to collaterally cleave surrounding rRNA. When comparing to the negative control (lane 2) in Figure 5, it is seen that all rRNA is degraded to some level in all other lanes. The rRNA in the positive control (lane 1) is totally degraded thereby leaving no signal. For the samples with Cas13a, sgRNA and target it is observed that the more time the samples were incubated the more rRNA is degraded. This indicates that the prolonged time gives Cas13a more time to degrade rRNA or that the rRNA is degraded because of the heat. Within the different time intervals, it is generally observed that the rRNA in the samples with 0,043 ng/µL sgRNA are most degraded ones. However, it is seen that the 0,0043 ng/µL samples are the less degraded ones besides from the negative control. This indicates, that for Cas13a to be fully activated an amount greater than 0,0043 ng/µL is needed. Thus, Cas13a and sgRNA formes a complex and is activated in the presence of the target. Thus indicating that the target activates Cas13a and thus works as expected.

Figure 1. Proof of Cas13a collateral cleavage being dependent on sgRNA concentration. All samples contain 20 ng/µL rRNA as a marker. Lane 1 and 2 are the positive and negative controls receptively. The positive control contains 1 µg/µL RNase A and the negative only contains rRNA and nuclease-free water. Lane 3-6 were incubated in 15 mins at 37°C, lane 7-19 were incubated for 40 mins and lane 11-14 were incubated for 3 hours. For each time samples with four different sgRNA concentrations were used. Thus, the samples contained 35 ng/µL Cas13a, 0.43 ng/µL target and 1.7 ng/µL. The varying concentration of sgRNA are lane 3, 7, and 11, = 0.43 ng/µL for lane 4, 8, and 12 = 0.043 ng/µL for lane 5, 9, and 13, = for lane 6, 10, and 14 = 0.0043 ng/µL.

Figure 2 shows that sgRNA-Cas13a complex is activated upon binding of the target sequence. The experiment is set up as the experiment in Figure 3 with sgRNA (BBa_K3602004) and target (BBa_K3602007). When the target was added to the samples, they were incubated in 40 mins. The experiment in Figure 1 showed that upon addition of target RNA to the sgRNA-Cas complex solution, collateral cleavage was induced. This can be observed as a smear in lane 5 and 6 thereby indicating degradation of rRNA. It is possible to see some degraded rRNA in all samples.

Figure 2. Proof of Cas13a collateral cleavage being dependent on sgRNA concentration. All samples contain 20 ng/µL rRNA as a marker. Lane 1 and 2 are the positive and negative controls receptively. The positive control contains 1 µg/µL RNase A and the negative only contains rRNA and nuclease-free water. Lane 3-6 were incubated in 15 mins at 37°C, lane 7-19 were incubated for 40 mins and lane 11-14 were incubated for 3 hours. For each time samples with four different sgRNA concentrations were used. Thus, the samples contained 35 ng/µL Cas13a, 0.43 ng/µL target and 1.7 ng/µL. The varying concentration of sgRNA are lane 3, 7, and 11, = 0.43 ng/µL for lane 4, 8, and 12 = 0.043 ng/µL for lane 5, 9, and 13, = for lane 6, 10, and 14 = 0.0043 ng/µL.

With these two results, it is shown that the target sequence can activate the sgRNA-Cas13a complexes, and thereby this part work as expected.

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