3.1 Qualtitative assesment of Cas12a orthologs

To select a suitable Cas12a ortholog for cronstructing the Cas sstaple, three different orhtologs were ordered from Addgene: AsCas12a (#69982), LbCas12a (#69988), and MbCas12a (#115142).

We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and FANCF. For comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.

Figure 3: Preliminary T7 Endonuclease I testing of Cas12a orthologs. T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 functions as a benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and FANCF were targeted. Editing is indicated by an extra band compared to the negative control.

3.2 Quantitative comparison between AsCas12a and MbCas12a

Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish between the better editing ortholog.
To accurately quantify the editing efficiency, we concted a dual luciferase assay. This assay measures the luminescence of Firefly luciferase, which decreases proportionally to the editing efficiency at the target site. To account for variations in cell count and transfection efficiency, the luminescence is normalized to Renilla luciferase, which acts as an internal control (Fig. 4). The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency compared to AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.

Figure 4: Comparison of AsCas12a and MbCas12a with a dual luciferae assay. Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group between the same Cas proteins are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

3.3 MbCas12a tolerates co-transfection and simultaneous editing of different Cas proteins

Wanting to employ the MbCas12a as part of a Cas staple, our goal was to find out how well MbCas12a can stay active while being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing 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.5 ).

Figure 5: Testing for Simultaneous Editing with Double Cut Luciferae Assay Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed contains MbCas12a and SpCas9. Cas12a 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

For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected, but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.

3.4 MbCas12a shows editing with fgRNA

To further confirm if MbCas12a is compatible with our Cas staples, editing rates were tested using a fusion guide RNA (fgRNA, BBa_K5237000) targeting two different loci: 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. 6). 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 MbCas12a editing utilizing a fgRNA.

Figure 6: 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. 7).

Figure 7: 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.

3.5 MbCas12a tolerates fusion to SpCas9

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 MbCas12a can stay active while being fused to SpCas9. 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. 8).

Figure 8: 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

For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a, strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.

3.6 MbCas12a fused to SpCas9 editing utilizing a fgRNA

The capability of MbCas12 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. We included biological duplicates in this assay.
MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they resulted in a higher editing efficiency than FANCF.

Figure 9: Editing rates for fusion guide RNAs with fusion Cas proteins. 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. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked as individual dots.

4.1 dMbCas12a Transactivation as Part of Cas Staple

The next step was to use the MbCas12a 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. 10 A). Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2. Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10 B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.

Figure 10: 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.