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Cas12a protein
purification crRNA synthesis Conjugation of magnetic
beads and ssDNA

Cas12a protein purification

Introduction

In our project, we combined three different biological reactions to detect an ASFV-specific DNA sequence in samples. The first is the LbCas12a-crRNA system, which can specifically recognize the ASFV-specific double stranded DNA (dsDNA) sequence of the p72 gene. The second reaction is the trans-activation of the LbCas12a-crRNA system. When the LbCas12a-crRNA system binds to ASFV-specific dsDNA sequences, the LbCas12a-crRNA system will not only cleave the dsDNA but also degrade non-specific single stranded DNA (ssDNA). To detect the degradation of ssDNA in the ASFV-activated LbCas12a-crRNA system, we will use the PicoGreen fluorescent dye to monitor the undegraded ssDNA, which is the third reaction. To allow detection of the reactions, we plan to conjugate ssDNA on magnetic beads. The ssDNA conjugated to magnetic beads will be easily captured and transferred by an electromagnet. In the following result section, we will show our progress through experiments that supported our project design.

Our targets

Steps to establish CRISPR-LbCas12a system

Expression of LbCas12a protein:

We transformed pHMT-LbCas12a into E. coli BL21, then added 0.2 mM IPTG to induce protein expression (see notebook for details). The result showed that the LbCas12a protein expression in soluble fraction was induced by IPTG, and increased as time went on (Figure 1). The predicted protein size of LbCas12a with MBP and His-tag is about 180 kDa, which is close to the induced protein indicated by the red arrow in Figure 1. We also examined the insoluble fraction of IPTG induced BL21 by SDS-PAGE, and confirmed that most LbCas12a protein was soluble (Figure 2)

Figure. 1. Coomassie Blue staining of total protein expression in supernatant of BL21 lysate at different time points.

Lane 1: Protein Marker (SMOBIO PM2600); lane 2: supernatant of BL21 without IPTG induction; lane 3 ~ 10: supernatant of BL21 induced by 0.2 mM IPTG at 16 °C for 16 hr.

Figure. 2. Coomassie Blue staining of total protein expression in pellet of BL21 lysate at different time points.

Lane 1: Protein Marker (SMOBIO PM2600); lane 2: pellet of cell lysate without IPTG induction; lane 3 ~ 10: pellet of cell lysate induced by 0.2 mM IPTG at 16 °C for 16 hr.

Pre-test of LbCas12a protein purification:

After confirming the induction of LbCas12a protein expression, we purified LbCas12a protein using Ni²⁺-magnetic beads to pull the His-tag on LbCas12a protein from the soluble fraction. Elution of LbCas12a protein from the Ni²⁺-magnetic beads by excess imidazole or TEV enzyme digestion further confirmed that the Ni²⁺-magnetic beads purification is clear and easy to reverse (Figure 3).

Figure. 3. Coomassie Blue staining of protein purification by Ni²⁺-magnet beads.

Lysate: supernatant of cell lysate; flow-through: supernatant before resuspension with Ni²⁺-magnet beads; TEV treated: treatment of Ni²⁺-magnet bead-LbCas12a complex by TEV enzyme.

According to the experiments above, we can express and purify LbCas12a protein for further applications. Therefore we scaled up the expression and purification of LbCas12a protein.

Large scale protein purification:

We use immobilized metal affinity chromatography (IMAC) to purify LbCas12a protein from the soluble fraction of BL21 in a Ni²⁺ chelating sepharose column. We then eluted LbCas12a protein from the Ni²⁺ column with imidazole and subjected it to FPLC separation. The absorption peak of fractions 27-30 is indicated by the red arrow in Figure 4.
To further confirm the absorption peak, we performed SDS-PAGE and Coomassie Blue staining of fractions 27-30, showing that the absorption peak is indeed LbCas12a protein, as shown in Figure 5.

Fig. 4. Chromatogram of LbCas12a purification by Ni²⁺ chelating sepharose column.

The numbers shown in red indicate the fractions. Each fraction was collected in 5 ml of wash or elution.

Figure. 5. Coomassie Blue staining of elution analyzed by SDS-PAGE.

M: marker; L: lysate; FT: flow-through; number: number of fraction tube.

crRNA synthesis

We performed PCR to amplify the DNA template of crRNA for in vitro transcription. The result, shown in Figure 6 showed the amplicon with correct size of 161 bp. However, we also observed multiplex amplicons with sizes around 400 bp and 600 bp. This suggested that we may need to reduce our template concentration in the PCR reaction.

Figure. 6. Gel electrophoresis of PCR product of crRNA template.

To generate a precise end to the transcription, we treated the DNA template of crRNA with KpnI restriction enzyme to remove any extra downstream sequence. The results are shown in Figure 8.

Figure 7. Gel electrophoresis of KpnI-treated DNA template of crRNA.

We then performed an in-vitro transcription assay to generate crRNA from the DNA template. We also performed gel electrophoresis to confirm the transcribed crRNA, as shown in Figure 8.

Figure 8. Gel electrophoresis of crRNA.

The right panel is an overexposure of the left panel.

Conjugation of magnetic beads and ssDNA

We developed a series of experiments to verify that the ssDNA had conjugated with magnetic beads.

UV-Visible spectroscopy:

First, we tested the UV-Visible spectroscopy of magnetic beads with and without the ssDNA. From the result, we found that the ssDNA with the magnetic beads shows up a peak around the absorption of O.D. 260 nm. The results imply that the magnetic beads had conjugated with the ssDNA.

Figure 1. UV-Visible spectroscopy of conjugated ssDNA

Dynamic Light Scattering

The zeta potential of the magnetic beads were tested. In Figure 10, ssDNA conjugated with magnetic beads shows a peak at the zeta potential of -23 mV, while magnetic beads without ssDNA shows the peak at -32 mV.

The Henry function below indicates that the particle radius is inverse proportion of the zeta potential under the same electrophoretic mobility. Base on this function, we confirmed that our magnetic beads had successfully conjugated with ssDNA.

Henry function:

Figure 2. Zeta potential from DLS testing

References

1. Kaszuba, M., Corbett, J., Watson, F. M., & Jones, A. (2010). High-concentration zeta potential measurements using light-scattering techniques. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 368(1927), 4439-4451.