2.1 The CRISPR/Cas System as a Gene Editing Tool

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 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 (RNP) 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. 1A). 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 RNAs (sgRNAs), 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 from the Cas9 gRNA and positioned on the 5' side of the crRNA (fig. 1B). 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 Dead Cas Proteins and their Application

Figure 3: Pre-fgRNA Maturation by Cas12a Depicted are the stages of a pre-fgRNA being expressed from the genome, cut by Cas12a into fgRNA molecules forming an RNP with the Cas12a.

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. Kweon et al.. (2017) further expanded the ways in which the CRISPR/Cas system could be used by introducing the concept of fusion guide RNAs (fgRNAs). By fusing the 3' end of a Cas12a crRNA 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, while allowing for Cas12a to process the pre-fgRNA into individual fgRNA molecules (Fig. 3). This allows for even greater multiplexing.

3. Assembly and Part Evolution

For the cloning, we employed the fgRNA entry vector (BBa_K5237000) resulting in a GGA using SapI with the insert being ordered as a DNA fragment.
Cloning via this strategy resulted in the designed and planned out construct being confirmed by Sanger sequencing (fig. 4)

Figure 4: Positive Cloning of the Desired Construct Confirmed by Sanger Sequencing. Two clones were picked and mini prepped after 16 h hours and sent to sequencing. Both clones had positive results and clean reads.

4. Results

Due to time constraints, we are not able to show data, nevertheless we are actively working on this assay.
The construct will be transfected into HEK293T cells together with a plasmid containing Cas12a and with a plasmid containing a fusion Cas (BBa_K5237003). The experiment will be carried out in technical replicates on a 6-well plate.
Lysis of the cells will occur 36 hours after transfection. Immediate RNA extraction will be performed with the miRNeasy Tissue/Cells Advanced Kit by QIAGEN to ensure the extraction of short RNA fragments below 200 nucleotides like the fgRNA.
When the extraction of the RNA was successful, reverse transcription into cDNA is started, followed by a qPCR. Each sample is screened with SYBR green labeled qPCR primers once for a housekeeper gene and for a sequence incorporated into the pre-fgRNA. Proper amplification between the chosen sites within the pre-fgRNA processing cassette can only take place when no processing has taken place into fgRNAs.

5. In Silico Characterization using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PICasSO system. DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA.
We calibrated DaVinci with literature and our own experimental affinity data obtained via EMSA assays and purified proteins. This enabled us to simulate enhancer hijacking in silico, providing valuable input for the design of further experiments. Additionally, we apply the same approach to our part collection.

DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged dna dynamics simulation. Here, we applied the last step to our parts, characterizing structure and dynamics of the dna-binding interaction.

5.1. Enhancer Hijacking is successfully studied in silico

Figure 11: Cas stapled plasmids.

With the Cas staple, we aimed to simulate the principles of enhancer hijacking experiments we conducted in the lab. For these experiments, we modeled the two plasmids also used in the wet lab (BBa_K5237023 and BBa_K5237024). On top of the two plasmids, we modeled the Cas staple by applying a “DNA-DNA tethering force” throughout our simulation, selectively on the regions targeted by the fgRNA. This force was based on simulation data acquired in earlier phases of DaVinci. As there is no suitable model available that also simulates proteins, this proved to be the most effective modeling strategy.

Our simulation showed the expected behavior, holding the target sequences of the Cas staple (Fig. 12). Overall, these exciting results demonstrate that we can successfully model the core principles of enhancer hijacking with a total of 20 thousand simulated nucleotides in silico.

Figure 12: Fusion Cas staples hold stapled nucleotides in close proximity.

5.2. Cas staple forces do not distrub DNA strand integrity

Figure 13: Cas stapled plasmids.

Next, we aimed to stress test our system to determine the amount of force required to induce DNA double-strand breaks. To achieve this, we used an identical setup to the previous experiment but instead of experimentally determined forces, we used artificial forces of varying strength. It is important to know that our in silico model responds to forces that cause double-strand breaks by scattering the nucleotides across the simulation box. As the specified bonds cannot actually break within the simulation, the phosphate backbone bonds become severely distorted and exhibit erratic behavior in the simulation videos. From our extensive simulations, we concluded that the forces required to damage the DNA are approximately 320 times (Fig. 15) and 680 times greater (Fig. 16) than the forces exerted by the Cas staple, respectively.

This provides important evidence regarding the safety of our staple approach for 3D genome engineering: According to our model, DNA-DNA tethering with our Cas staples is not expected to have a negative effect on the DNA stability.

Figure 14: Applying a force that is 270 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 15: Applying a force that is 320 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 16: Applying a force that is 680 times greater
than the predicted force typically exerted by a Cas staple on DNA.

Figure 17: Applying a force that is more than 1000 times greater
than the predicted force typically exerted by a Cas staple on DNA.

5.3. DaVinci Helps to Design Multi-Staple Arrangements

Figure 18: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.

Finally, we simulated the behavior of multiple staples at once. Therefore, we expanded our previously introduced experimental setup by a second Cas staple.
In a first approach, we targeted an additional region next to the original chosen one. This region is 40 nucleotides away from the first target region on the plasmid displayed in blue connecting it to the opposite site of the orange plasmid, therefore bringing both ends of the orange plasmid together (Fig. 18).
Our simulation predicted that with these staple points, DNA double-strand breaks would occur as clearly visible by the scattered nucleotides (Fig. 19).

Figure 19: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.

Figure 20: Stable multiplexing with 2 Cas staples. On the blue plasmid, the Cas binding sequences are 980 nucleotides apart.

To simulate a setup where we expect no double-strand breaks, we increased the distance between the stapling sites on the blue plasmid (BBa_K5237023) from 40 to 980 nucleotides (Fig. 20).
With this increased distance between stapling sites, we observed a stabilized system. Most interestingly, the non-stapled regions showed maximum distances close to 500 nm, indicating that the two staples led to more compact plasmid structures.

In conclusion, we show that applying multiple staples on the same structures can lead to double-strand breaks if the staples are positioned closely to one another. However, increasing the separation of staples leads to a stable system without DNA damage. Thus, it is definitely possible to multiplex Cas staples, thereby increasing the compactness of DNA structures (Fig. 21). This shows the potential of creating complex regulatory networks by bringing genetic elements into spatial proximity with a multitude of Cas protein staples.

Figure 21: Increasing the distance between Cas staple targets to 980 nucleotides stabilizes multiplexing.

5. References

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Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. Cell 187, 1076–1100. https://doi.org/10.1016/j.cell.2024.01.042.

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