Part:BBa_K3602015:Experience

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Applications of BBa_K3602015

Protein purification and His-tag removal

The His-tags are seen to work. This is seen in Figure 1 as the Cas12a protein was successfully purified from E. coli K12 using His-tag purification.

Figure 1. Successful protein purification of Cas12a. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas12a+SUMO-Histag = 156,31 kDa. The first lane displays a sample of lysed E. coli ‘ER2536 + Cas12a plasmid’ flow-through before induction of IPTG. The second lane displays a sample of lysed E. coli ‘ER2536 + Cas12a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The fourth lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lanes 7-12 display 1 mL elution-buffer fractions.

Cas12a function

The function of Cas12a being an active endonuclease when in complex with a sgRNA and in the presence of a target sequence are supported by the following experiment. In the experiment, Cas12a was used to detect the target part BBa_K3602012 with the help of the matching sgRNA to induce cleavage of an ssDNA reporter shown in an agarose gel electrophoresis.

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 Cas12a can form a complex with sgRNA and that in the presence of a target sequence it is activated and starts collateral cleavage.

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.

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