Part:BBa_K3602012:Experience

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Applications of BBa_K3602012

rs16901979-A SNP activates Cas12a

The activation of Cas12a by the rs16901979-A target is supported by the following experiment. In the experiment, Cas12a (BBa_K3602022) was used to detect the target with the help of the matching sgRNA to induce cleavage of an ssDNA reporter shown in an agarose gel electrophoresis.

Figure 2. Cas12a activity on 2% agarose gel. All samples were tested on the rs16901979 C;A genotype and received the same amounts of target and reporter DNA. Lanes 1-4 contain the controls used, where double-stranded Cas13a was either treated with DNAse or not and was digested on an EcoRI restriction site and thereafter boiled at 95°C to open the strand. Lanes 5-8 show the samples of either ds- or ssDNA that received 1 µM sgRNA+target and lanes 9-13 contain samples that received 2µM sgRNA+target. Lastly, lanes 13-16 show bands for samples that did not receive any target sequence yet received 2 µM sgRNA. The results of the gel show that Cas12a is actively degrading the reporter DNA in samples that used ssDNA and received 2 µM sgRNA+target. This is confirmed by the band exhibited in lane 13, where Cas12a was not active due to the absence of a target sequence.

It can be observed in lanes 9-12 that Cas12a was only activated when paired with ssDNA, 2µM sgRNA and target DNA. No activity can be seen with only 1 µM concentration of gRNA. A negative control was made with 2µM sgRNA found on lanes 13 and 16, Cas12a and no target which showed no change in ssDNA concentration from the negative control with the only ssDNA.

Furthermore, digested DNA that was not subjected to boiling (lane 3) showed that the linearization of the plasmid was successful. Through the ssDNA, we were also able to observe unspecific activity in which Cas12a can cut dsDNA, but only if there is a match between the gRNA and dsDNA sequence. This was not the case for our reporter. Lastly, no digestion of plasmid was observed in lanes 13-16, indicating that there were no off-target effects from Cas12a.

It is thereby shown that this target sequence can activate sgRNA-Cas12a complexes, thereby working as expected.

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