2.1 Discovery and Mechanism of CRISPR/Cas9

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. 2 A). 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).

Figure 2: The CRISPR/Cas system (adapted from Pacesa et al. (2024)) 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

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. 2 B). 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 5nt long (Paul and Montoya, 2020).

2.3 Dead Cas 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

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).

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 guidingactive Cas12a and Cas9 to two distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator towards a target locus.

4.1 Editing endogenous loci with fgRNAs

To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the Cas protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I assay.
AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The samples included standard single gRNAs with the corresponding Cas protein, the fgRNA with only one of the two Cas proteins and the fgRNA with both Cas proteins simultaneously (Fig. 5). The sgRNAs allowed for the highest editing rates for both genes (45% for VEGFA and 15% for FANCF), while the editing rates for FANCF were consistently lower in all experiments. Importantly, targeting FANCF with fgRNAs resulted in noticeable editing of about 10%, with just the SpCas9 and both Cas proteins in the sample. For VEGFA, the AsCas12a only sample resulted in approximately 20% editing rate in combination with the fgRNA, while adding both Cas proteins led to approximately 40%. These initial results confirmed our engineering approach proving efficient genome editing with fgRNAs.

Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci. 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.

4.2 Fusion Guide RNAs Allow for Editing Rates With Variant Cas Orthologs

To further evaluate the capabilities of the fgRNAs, we tested them in combination with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9, because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared to MbCas12a (Fig. 6). Between these two co-transfections the SpCas9 editing has not been significantly different.

Figure 6: 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

Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting FANCF and SpCas9 targeting VEGFA and once vice versa. 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. 7). 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. When targeting the same gene under the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.

Figure 7: 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.

4.3 The Inclusion of a Linker Does Not Lower Editing Rates

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. 8). 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.

Figure 8: 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.

4.4 fgRNAs can be used for CRISPRa

To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the use of fgRNAs for CRISPR activation. For this, we intend to recruit the transcriptional activator VP64 to a firefly luciferase gene to induce expression. The VP64 protein is attached to the catalytically inactive Cas9 protein, which is then guided by gRNAs to the luciferase gene. The gRNAs target a TetO sequence, which is positioned in front of the luciferase gene in multiple repeats. The firefly luciferase activity was then quantified as photon counts and normalized against Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous promoter. In two biological replicates we saw similar Relative luciferase activity with fgRNA as a guide compared to a sgRNA (Fig. 9).

Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. The tetO repeats were targeted by Cas9-VP64, once with a sgRNA and once with a fgRNA that had a non-targeting sequence for the Cas12a spacer. 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.

4.5 Stapling Two DNA Strands Together Using fgRNAs

After showing the general capability of the fgRNA to work for editing and for CRISPR activation, the next step was to use it 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, Panel 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. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene. These results suggest an extension of the linker might lead to better transactivation when hijacking an enhancer/activator.

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.