2.1 Discovery and Mechanism of CRISPR/Cas9

Figure 2: The CRISPR/Cas System A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with their respective PAMs. The sgRNA/crRNA spacer sequence binds the DNA target strand via complementary base pairing. 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 be bound by the Cas protein. DNA cleavage sites are indicated 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 in a programmable manner. 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, an RNA guide is complexed by multiple Cas proteins, whereas class 2 systems consist of a singular protein binding 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 sequence 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) at the 3' end in the targeted DNA is needed for Cas9 DNA 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 the CRISPR/Cas9 system was the introduction of single guide RNAs (sgRNA[s]), which combine the functions of a tracrRNA and crRNA (Jinek et al., 2012; Mali et al., 2013). Moreover, Cong et al. (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, several additional class 2 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. In contrast to the type II systems, the Cas12a RNA guide only requires a crRNA to mediate Cas12a DNA targeting. Moreover, Cas12a is capable of processing long precursor crRNA transcripts into several, single/independent crRNAs, 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 Cas protein mutants that retain their DNA binding capability, but have no catalytic activity (Koonin et al., 2023) (Kleinstiver et al., 2019). The latter 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 genes via complementary spacer sequences (Kampmann, 2017). A common approach for CRISPRa involves fusing Cas9 with the transcriptional activator, such as VP64 or VPR (Kampmann, 2017).

4.1 Editing endogenous loci with fgRNAs

To show that our fusion gRNA design results in an active CRISPR-Cas ribonucleoprotein complex, a series of different fgRNAs were cloned, each carrying spacer sequences specific to the VEGFA and FANCF target genes. HEK293T cells were then co-transfected with the Cas protein and (f)gRNA encoding constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I assay.
Here, AsCas12a and SpCas9 were used. The AsCas12a spacer, in this case, targets VEGFA, while the SpCas9 spacer targets FANCF. Samples included either (i) conventional single gRNAs co-expressed with the corresponding Cas proteins (positive controls), (ii) the fgRNA co-expressed with only one of the two Cas proteins (as control for Cas ortholog dependency) and (iii) the fgRNA with both Cas proteins simultaneously co-expressed (Fig. 5). The conventional sgRNAs resulted in potent editing ("editing" refers to the observed indel frequency) for both target genes (45% for VEGFA and 15% for FANCF). Note that editing rates for FANCF were consistently lower in all experiments, which likely is due to the specific properties of the FANCF-targeted locus. Importantly, as hoped, targeting FANCF with fgRNAs resulted in noticeable editing of about 10%, when we added the SpCas9 alone or both Cas proteins into the sample. For VEGFA, the AsCas12a only sample resulted in approximately 20% editing in combination with the fgRNA, while adding both Cas proteins led to approximately 40%. These initial results confirmed that our fgRNA design indeed functions, enabling simultaneous recuitment of two different Cas proteins to separate loci in human cells.

Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci. Editing rates were asessed 72h post transfection via T7EI assay. Editing % was determined by measuring intensities of T7 cleavage bands vs. the input band in ImageJ; Editing % was then calculated as follows: Editing % = 100 x (1 - (1- cleaved band/uncleaved band))^1/2. The schematics at the top shows the composition of the fgRNA. Below each spacer (cartoon), the targeted gene is indicated. The symbols below indicate which parts are included in each sample.

4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs

After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs. To this end, we tested fgRNAs in combination with different Cas12a orthologs. Following some initial testing, we decided to use MbCas12a together with SpCas9 in subsequent experiments, since MbCas12a turned out to be more effective than AsCas12a in a dual luciferase assay when co-transfected with SpCas9 (Fig. 6). Of note, SpCas9 reproducibly showed high potency.

Figure 6: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Reporter Assay. HEK293T cells were co-transfected with the indicated constructs and firefly and Renilla luminescence intensity was measured 48 h after transfection. Relative luciferase units correspond to firefly luciferase photon counts normalized to Renilla luciferase photon counts (Renilla luciferase serves as transfection control). Potent activity of CRISPR-Cas effectors result in knockdown of the firefly luciferase and hence smaller values. Samples correspond to cells co-transfected with a firefly luciferase targeting fgRNA and the constructs encoding the indicated CRISPR-Cas effector (see color legend). Data represent the mean +/- SD from n = 3 independent experiments. Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. ***p<0.001,

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 Fusion Guide RNAs 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 CRISPRa. 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.