2.1 Discovery and Mechanism of CRISPR/Cas9

Figure 2: The CRISPR/Cas System Overview A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM. The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a specific secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are symbolized by the scissors

In 2012, Jinek et al. discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein complexes with Cas9 (Pacesa et al., 2024). They include a CRISPR RNA (crRNA), which specifies the target with a 20 nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the Cas protein (Jinek et al., 2012) (Fig. 2A). Furthermore, a specific three nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred to as the protospacer adjacent motif (PAM) (Sternberg et al., 2014). The most commonly used Cas9 protein is SpCas9 or SpyCas9, which originates from Streptococcus pyogenes (Pacesa et al., 2024).

A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the functions of a tracrRNA and crRNA (Mali et al., 2013). Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence accordingly.

2.2 Differences Between Cas9 and Cas12a

Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has been classified as Cas12a since then (Zetsche et al., 2015). Cas12a forms a class 2 type V system with its RNA, that in comparison to the type II systems, only requires a crRNA for targeting and activation. Cas12a is capable of processing the precursor crRNA into crRNA independently, whereas Cas9 requires the RNase III enzyme and tracrRNA for this process (Paul and Montoya, 2020). This crRNA is often also referred to as a guide RNA (gRNA). However, the stem loop that is formed when binding the Cas protein is structurally distinct to the Cas9 gRNA and positioned on the 5' side of the crRNA (Fig. 2B). Similarly, the PAM (TTTV) is also on the 5' side (Pacesa et al., 2024). Cas9 possesses RuvC and HNH domains that are catalytically active, each of which cleaves one of the DNA strands at the same site, resulting in the formation of blunt end cuts (Nishimasu et al., 2014). Cas12a possesses one RuvC-like domain that creates staggered cuts with overhangs that are about 5 nt long (Paul and Montoya, 2020).

2.3 dCas Proteins and their Application

Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin et al., 2023) (Kleinstiver et al., 2019). These are referred to as dead Cas proteins or dCas9 and dCas12a. These Cas proteins can be used to activate (CRISPRa) or inhibit (CRISPRi) the expression of genes by fusing them to effector domains and targeting the respective gene with the spacer sequence (Kampmann, 2017). A common approach for CRISPRa involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017).

2.4 fgRNA and CHyMErA System

Figure 3: Applications of the Fusion Guide RNA (adapted from Kweon et al. (2017)). Fusion Guide RNAs can be used for multiplex genome editing by guiding active Cas12a and Cas9 to two distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator towards a target locus.

Kweon et al. (2017) further expanded the ways in which the CRISPR/Cas system could be used by introducing the concept of fusion guide RNA (fgRNA)s. By fusing the 3' end of a Cas12a gRNA to the 5' end of a Cas9 gRNA, the newly created fgRNA could be used by both proteins independently for either multiplex genome editing or transcriptional regulation and genome editing in parallel (Fig. 3). Similarly, this is also possible using the Cas Hybrid for Multiplexed Editing and screening applications (CHyMErA) system (Gonatopoulos-Pournatzis et al., 2020). In this instance, the gRNAs of Cas12a and Cas9 are connected in the opposite direction (3' Cas9 gRNA to 5' Cas12a gRNA), allowing for Cas12a to process the RNA into individual units (Fig. 3). Amongst other things, this allows for the analysis of the interaction between different genes by targeting them simultaneously with the two distinct spacers (Aregger et al., 2021) (Fig. 3).

Building on the CRISPR/Cas system's versatile functionality in genome editing, recent advances have extended its applications into DNA nanotechnology. Traditionally, DNA nanostructures have been constructed through the hybridization of multiple single-stranded DNAs. However, a new strategy leverages the CRISPR system to create double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructures. Using the dCas proteins, which were previously described for their gene regulation capabilities (Wu et al.(2022)). Once the RNP has formed, the bivalent fusion dCas can precisely recognize target sequences on a double-stranded DNA, pulling them together to form intricate hybrid nanostructures. These nanostructures resemble DNA-protein hybrids found in chromosomes, mimicking the genomic structure and enabling stimuli-responsive gene regulation. This innovative use of dCas proteins not only extends the capabilities of the CRISPR system but also presents new opportunities for advancing DNA nanotechnology.

3.1 Decision of Cas Proteins for the Staple

As one part of our staple we decided on SpCas9, as it well characterized. Three different orthologs 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.
For the next assay we constructed an AsCas12a fusion with SpCas9 and a MbCas12a fusion with SpCas9 by PCR amplifying the Cas12a orthologs out of the plasmids used in prior tests and cloning them into the plasmid containing the SpCas9.
To accurately quantify the editing efficiency, we conducted 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 Luciferase 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 grouped by fused proteins. Co-transformed contains single Cas proteins, in contrast to the fusion Cas having the same cas proteins covalently bound to each other. 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 between the same Cas proteins are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

For the single co-transfected Cas proteins the SpCas9 showed, as expected, the highest editing efficiency, shown by low relative luminescence units. Contradicting the T7EI, but being in-line with the previous dual luciferase, MbCas12a showed better editing efficiency compared to AsCas12a (p=0.005). The Fusion Cas proteins exhibited less editing efficiency compared to the single counterparts. Of the MbCas12a-SpCas9 fusion, SpCas9 showed higher efficiency. For the Cas12s in the fusion Cas, both proteins exhibit low editing activity, with no significant difference. These results left us with the conclusion to further pursue MbCas12a for fusion Cas construction. Further testing of the catalytically active version is needed before cloning the dMbCas12a variant.

3.3 MbCas12a and SpCas9 Remain Functional after Fusion

Testing showed simultaneous editing of both 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. Same results could be observed for SpCas9 with the single cut showing highly significant editing. When introducing targeting gRNAs for both Cas proteins we see no reduction in the highly significant editing compared to only one targeting, strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.

3.4 Combining Fusion Cas Editing with fgRNA

The capability of 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 Forming a Cas Staple with Fusion Cas and fgRNA

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 dual luciferase assay was used, with one enhancer plasmid and one reporter plasmid. The fusion Cas proteins can be used to increase expression levels of the reporter firefly luciferase (Fig. 12). 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 10: Reporter Trans Activation through Fusion Cas and fgRNA In A and B 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.