3.1 SpCas9 can be Co-Transfected wWth other Cas Proteins

Wanting to employ the SpCas9 as part of a Cas staple, our goal was to find out how well SpCas9 can stay active while being transfected together with MbCas12a. Therefore we engineered the dual luciferase assay to allow us to test two catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla luciferase gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 3).

Figure 3: Double cut dual luciferase assay testing Fusion Cas simultaneous editing. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

3.2 SpCas9 shows editing with fgRNA

Wanting to test if the different parts of our Cas staples will work together, we tested editing rates of SpCas9 using fgRNA. Two spacers were tested: FANCF and VEGFA. To better assess the impact that the utilization of a fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.
Having the sgRNA with single Cas proteins in the same sample resulted in no clear difference in the editing rates (Fig. 4). The fusion of the gRNAs resulted in a lower editing rate overall. While the editing for VEGFA stayed at about 20% in all cases, the editing for FANCF dropped significantly. Nonetheless we were able to show SpCas9 editing utilizing a fgRNA.

Figure 4: Fusion gRNA Editing Rates In Combination with MbCas12a. In A and B the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. A and B display both orientations of the two spacers for VEGFA and FANCF.

To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 5).

Figure 5: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA. The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) ^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5.

For CCR5, the editing rate with sgRNAs was approximately the same at about 30%. However, it dropped below 10% for the fgRNA. The addition of the 20 nt linker had no effect on the editing rates compared to no linker.

3.3 SpCas9 can be fused to MbCas12a while maintaining functionality

Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion, potentially giving us another way of Cas stapling. Our goal was to find out how well SpCas9 can stay active while being fused to MbCas12a. Therefore we employed the previously engineered dual luciferase assay to allow us for testing two catalytically active Cas proteins at once, this time being fused to each other (Fig. 6).

Figure 6: Double cut dual luciferase assay testing Fusion Cas simultaneous editing. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

MbCas12a showed highly significant editing rate in the single cut experiment (only MbCas12a has a targeting gRNA). When introducing a targeting gRNA for SpCas9 no significant change could be detected, strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.

3.4 SpCas9 fused to MbCas12a shows editing with fgRNA

The capability of SpCas9 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this, the same target sequences as before were used, namely FANCF and VEGFA in both configurations. Biological duplicates were done for this assay.
SpCas9 editing rates were higher overall. At the same time, when targeting VEGFA they resulted in a higher editing efficiency than FANCF.

Figure 7: Editing rates for fusion guide RNAs with fusion Cas proteins. the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) ^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked as individual dots

4.1 dSpCas9 transactivation as part of a Cas staple

The next step was to use the SpCas9 as part of a Cas staple to staple two DNA loci together, and thereby induce proximity between two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 8, A). Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 8, B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.

Figure 8: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

4.2 SpCas9 fused to dMbCas12a form the Cas staple

Continuing the procedure in a similar manner, we focused on inducing proximity between genetic loci using the fusion dCas next, consisting of dMbCas12a fused to dSpCas9. The same assay was used, with one enhancer plasmid and one reporter plasmid. Though less distinct than the results for not using a protein fusion, the fusion Cas proteins can be used to increase expression levels of the reporter firefly luciferase (see figure 13). While using sgRNAs results in similar relative luciferase activity as for the negative control between 0.1 and 0.2., using a fgRNA with a 20 to 30 nt linker consistently resulted in activities at 0.25. Fusion guide RNAs without a linker and with a 40 nt linker had on average about the same activity, but with a higher spread over the biological replicates. Nonetheless this showed the dMbCas12a fused to the dSpCas9 to be working as a Cas staple. Further tweaking is needed to get better results

Figure 9: Firefly luciferase trans activation through fusion Cas staple Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). Fusion Cas proteins were paired with sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt