Results

After isolating plasmid of interest (pASK-IBA2) from E. coli DH5α, we linearized it by PCR using appropriate primers.

Gibson assembly (see “Gibson assembly” in protocols under cloning and transformation) was performed to insert our construct coding-sequences into the linearized vector. We succeeded in creating HLC and CLS coding-sequence-containing plasmids.

E.coli Top10 cells were transformed with our plasmids, and single-colony PCR with primers targeting insertion site was performed at the same time as colonies were inoculated in liquid culture. The gel shown in Figures 1 and 2 indicates the successful insertion of gBlock sequences, as seen by the bands corresponding to ̴ 1500 kB. The results of the gels were later confirmed by Sanger sequencing of the isolated plasmids.

E.coli BL21 (DE3) Gold cells were then transformed with the plasmids which had no mutations deemed to be harmful to expression. After transformation single colony PCR was performed, the result of which is seen in Figure 3 where bands of expected size ̴ 1500 kB are present. The bands seen in figure 3 (wells 2 through 9, with well 1 containing the ladder) seem to deviate slightly in size, this could potentially have been caused by the gel itself not being perfectly cast.

One of each colony for HLC and CLS were chosen. Glycerol stocks were made and kept at -80°C for future experiments.

After confirming that BL21 single colonies were transformed with our plasmids (se figure 3) we continued with small-scale expression to figure out optimal conditions. At first, we noticed that most of our protein ended up in the insoluble fraction (see figure 4).

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BL21 (DE3) Gold cells were then transformed with the plasmids which had no or few mutations deemed to be harmful to expression. After transformation single colony PCR was performed, the result of which is seen in Figure 3 where bands of expected size ̴ 1500 kB are present. The bands seen in figure 3 (wells 2 through 9, with well 1 containing the ladder) seem to deviate slightly in size, this could potentially have been caused by the gel itself not being perfectly cast.

After confirming that BL21 single colonies were transformed with our plasmids (se figure 3) we continued with small scale expression to figure out optimal conditions. At first, we noticed that most of our protein ended up in the insoluble fraction (figure 9).

Due to both HLC and CLS being tagged variants of dCasMINI (a modified cas12f), we decided to test 16 °C overnight and room temperature both of which gave large amounts of CLS in the soluble fraction. We decided to test if the different tags between the 2 proteins would affect their optimal expression condition. After testing both conditions, we determined that the highest amount of HLC in the soluble fraction was 16 °C overnight (figure 10).

After successfully producing CLS on a larger scale (2x1L) we aimed to purify the protein using a StrepTrap XT column packed with Strep-Tactin® XT Sepharose resin to purify our Strep-tagged CLS protein [3]. The chromatogram in figure 11 shows a single large peak after the isocratic point which indicates the addition of buffer e (see buffers). For affinity chromatography with this specific column, this is likely to be the point where our target protein was eluted. The rather slow drop in the UV signal indicates that not all of the CLS protein was eluted simultaneously and that there are other proteins that were bound to the column. This was confirmed by SDS gel of the fractions produced (see figure 12).

Several fractions were run on a gel to confirm which fractions could be combined and further concentrated. Figure 12 shows that fractions F11 to F14 could be combined and concentrated with a 20 ml ~30 kDa molecular weight cut off filtration filter tube (vivaspin centrifugal concentrators). The end protein sample concentration was 39.6 µM. This is enough for a stock amount to then later be used in EMSA – protein/sgRNA binding assays. As can be seen in figure 13 the band corresponding to ~64 kDa has decreased in proportional size and intensity, whilst multiple other bands, particularly smaller bands are gone or weaker relative to the ~64 kDa band. The presence of smaller bands indicates that either CLS is being degraded or that the concentration was done improperly. Whilst the decrease in the intensity of the 64 kDa can be due to limitations in the taken sample or improper concentration.

After successfully producing HLC on a larger scale (2x1L cultures) we aimed to purify the protein using a HisTag FF column. The chromatography results of the His-tag purification show one peak (figure 14) that likely corresponds to eluted HLC. To not lose most protein and confirm which fractions contain HLC we ran an SDS-PAGE gel. According to figure 15 fractions 9 to 13 were eligible to be combined and concentrated, which resulted in 22.22 µM of HLC. This is enough stock amount to have later be used in EMSA – protein/sgRNA binding assays and protein/sgRNA/DNA binding assays.

For affinity chromatography with this specific column, this is likely to be the point where our target protein was eluted. The rather slow drop in the UV signal indicates that not all of the CLS was eluted simultaneously and that there are other proteins that were bound to the column. This was confirmed by SDS gel of the fractions produced (figure 15).

Several fractions were run on a gel to confirm which fractions could be combined and further concentrated. Figure 15 shows that fractions F9 to F13 could be combined and concentrated with a 20 ml ~30 kDa molecular weight cut off filtration filter tube (vivaspin centrifugal concentrators). The end protein sample concentration was 22.22 µM. This is enough for a stock amount to then later be used in EMSA – protein/sgRNA binding assays. As can be seen in figure 16 the band corresponding to ~64 kDa has increased in proportional size and multiple other bands, particularly smaller bands are gone or weaker relative to the ~64 kDa band. The presence of smaller bands indicates that either HLC is being degraded or that the concentration was done improperly .

sgRNA synthesis was done using the New England BioLabs T7 hiscribe in vitro transcription kit according to the Standard RNA Synthesis (E2040) protocol (new England biolabs). The reaction was incubated for 4 hours before the RNA was treated with DNase 1 (DNase1 see protocols), and the reaction was purified in a Qiagen RNeasy clean-up mini kit. The resulting RNA sample was then analyzed with a nanodrop to estimate concentration, and with a bioanalyser using a total RNA nano kit to estimate RNA quality.

The sgRNA1 gave a concentration of 35,4 ng/ul and showed some peaks for shorter than the intended sgRNA size during bioanalyzer analysis (se figure 15).

The sgRNA2 gave a concentration of 33,2 ng/ul and showed some peaks for shorter than the intended sgRNA size during bioanalyzer analysis (figure 16).

To test the binding of our sgRNAs to the dCasMINI proteins, we decided to perform an EMSA (electrophoretic mobility shift assays) experiment which would allow easy visualization of the binding by the change in band position for our RNA and/or protein (depending on the staining method). As Xu et.al only worked with cell cultures, and our test intended to be done in an in vitro non-cellular environment. We found it necessary to establish the optimal conditions for this reaction. As such we based our original method on the work of Li et.al who worked with the structure of “apo Lb2Cas12a”, a cas protein in the same family as Cas12f (and by extension dCasMINI). The reaction was conducted in the binding buffer (link here to RNA binding buffer), at 4°C for 15 minutes before being loaded on a gel.

First, we tried running Native gels to visualize our protein constructs binding to the sgRNA. Due to the sgRNA DNA template primers being delivered late, we had to work with a very small amount of our sgRNA. This influenced our reaction volume. We decided to work with 100 nM sgRNA with 100 nM and 200 nM of protein in a 5 ul reaction. The total amount of protein in this reaction was not sufficient for the staining methods attempted (se figure11) for visualization of the protein.

We then decided to run a 2% agarose gel electrophoresis like that done in the protein RNA interaction work of Ream et.al. When the reaction was run on an agarose gel we were able to see weak bands for RNA in the expected size. This proved to us that dcasMINI did not bind to sgRNA under these conditions, since RNA bands were visible in all 3 loaded wells at the same length (Figure 20). This also demonstrated that our RNA was not degraded under these conditions.

We saw no binding in the starting conditions we decided to try to optimize. Seeing that Kupcinskaite et.al and Anders et.al used higher temperatures and longer reaction times, we decided to increase the incubation temperature and duration for our reaction. We then tested incubation at 37 °C for 25 minutes. The reaction was loaded on a 2% agarose gel to visualize a change in the migration of the sgRNA bands. Unfortunately, these changed conditions did not cause any binding to occur. This is seen by the lack of any bands of greater size than those seen in the control (see fig), a band of greater size would indicate that the RNA has been bound to something that slows its progress through the gel.

Experiments that did not succeed yet:

We have yet to optimize binding conditions for our guide RNAs and CasMINI proteins.

Future plans: