Introduction
Introduction
The CRISPR / Cas System as a Gene Editing Tool
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Strategies to Program DNA Conformation
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Aim of This Subproject
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Results
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Static Molecular Interaction Predictions
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Results
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All-Atom Dynamics Simulations
By developing the EMSA and FRET assays, we established key tools for the rational design and evaluation of various protein staples. Beginning with the successful construction of the basic staples, these assays provided key insight into DNA-binding proteins and stapling mechanisms. Through thorough testing of our workflow, we developed foundational techniques that future iGEMers and researchers can leverage to engineer and optimize protein-based DNA-folding systems.
Results
Long-Range DNA Dynamics Simulations
Results
We aimed to create a digital twin of the enhancer hijacking experiments we conducted in the lab. There, we used one plasmid containing a trans-activation domain and another containing a reporter gene.
By supplying the protein staple, we were able to show that expression of the reporter gene could be induced. Without a staple, the reporter gene was not expressed anymore. With our in silico
replica, we wanted to understand the stapling dynamics better to investigate potential effects on the bound DNA. To do so, we started by simulating two plasmids without a staple, generating a baseline to compare later results.
As seen in Figure 2, the distances between the proximity-induced nucleotides on two different plasmids increased over time. This clearly shows that a staple is needed to keep the plasmids in proximity. Furthermore, all other nucleotides
showed a uniform distribution for all distances up to around 700 nm. This demonstrates that the ends of both plasmids are approximately 700 nm apart. With our gained knowledge, we started looking into the specific dynamics of enhancer hijacking.
Assumptions
- Stapling distances: Distances of the stapled nucleotides are predicted in our all-atom simulation, based on the Cas staple with a fgRNA containing a 40 nt linker
- Stapling forces are taken from atomistic predictions of what forces the fusion Cas protein staple exerts on its binding sequences
- For more details see Core Assumptions
Besides these two plasmids, we also added the Cas staple by applying its forces to our simulation. As mentioned in “all-atom dynamics simulations”, we predicted and simulated the dynamics of our Cas staple (BBa_K5237003). We used the estimated distances at which the Cas staple holds the nucleotides targeted by the fgRNA. Additionally, we averaged the predicted forces exerted by the Cas staple onto these nucleotides. This created an accurate and precise model with an expected distance between the fgRNA-targeted nucleotides on both plasmids of 7.4 nm. With the predicted distances and forces, we started simulating the synthetic enhancer hijacking experiments conducted in the lab.
We found that our simulation accurately holds the target sequences of the Cas staple at the expected distance of 5-10 nanometers (see Figure 4, light blue histogram). As it is shown as a single peak, this means that no double-strand breaks occur - meaning that the forces exerted onto our DNA do not pose a safety hazard. The distance between all not-targeted nucleotides was uniformly distributed and showed that the ends of both plasmids are around 700 nm apart.
These results show that we can successfully model enhancer hijacking with a total of 20 thousand simulated nucleotides in silico .
Assumptions
- Stapling distances of stapled nucleotides is defined so that they adhere to a fully stretched and linear fgRNA with a 40 nt linker
- Stapling forces are an average from atomistic predictions of what fusion Cas protein staples exert on their binding sequence
- For more details see Core Assumptions
While there is a strong local peak around the expected distance for the stapled nucleotides in Figures 6C and 6D, the wide distribution with another maximum of around 400 nm indicates that the targeted nucleotides do not stay in close contact over the entire simulation time. 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. This means that as soon as the stapled nucleotides show more than one peak, DNA double-strand breaks occur. The forces needed to damage the DNA started for stiffnesses between 1.4 and 3 (see Figure 6) - an increase of 300 and 680 times compared to the actual Cas staple forces, respectively.
As expected, the reference nucleotides showed a relatively uniform distribution. When comparing the plots from Figure 6 with one another, the reference nucleotides represent the baseline accurately for different forces. Interestingly, the far ends of the plasmid are never further separated than 700 nm. This however is probably influenced by the simulation box, which prevents the nucleotides from reaching greater distances. In actual cells, the broken strands would disperse way further.
All in all, we concluded that the forces needed to damage the DNA integrity are more than 380 times stronger than those directed onto the DNA by the Cas staple (see Figure 6B). This proved the safety of our staple: Binding by Cas staple won’t have a direct negative effect on the DNA.
Assumptions
- Stapling distances of stapled nucleotides are defined so that they adhere to a fully stretched and linear fgRNA
- Stapling forces amount to the average force exerted by fusion Cas protein staples on their binding sequence during atomistic predictions
- For more details see Core Assumptions
Firstly, we targeted a sequence 40 nucleotides away from the first target sequence on the plasmid displayed in blue. The other target of the second staple was located on the opposite side of the plasmid shown in yellow (see Figure 7).
As shown in Figure 8, our simulation predicted that with these staple points, DNA double-strand breaks are induced. This is inferred from the scattering of targeted nucleotides across the simulation box and a missing single peak for the expected distance of 7.4 nm. While this expected distance still presents the global maximum for the distribution, the simulation did show that the distances between stapled nucleotides increased over time. From inspecting the simulation video, we learned that both plasmids started to break.
To avoid double-strand breaks, we increased the distance between the stapling sites on our first plasmid (BBa_K5237003) from 40 to 980 nucleotides (Fig. 9).
With this increased distance between stapling sites, we observed a stabilized system. Both staples showed a single strong peak for the expected distances of around 20 nm (see Figure 10) while the reference nucleotides were more widely spread out. Most interestingly, these 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. This shows the potential of creating complex regulatory networks by bringing genetic elements into spatial proximity with a multitude of Cas protein staples.
Discussion and the road ahead
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Introduction
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It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Road ahead
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Results
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Discussion
Editing Efficiency of fgRNAs Is Influenced by Numerous Factors
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
lorem text halt
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
Outlook
It has long been known that the three-dimensional organization of DNA has major implications in many contexts from plasmid delivery into cells over the transcriptional activity of genes to the spatial organization of chromosomes or whole genomes. Importantly, structural and orientational rearrangements of DNA are associated with several genetic diseases including cardiomyopathy and cancer (Dong et al., 2023) (Watanabe et al., 2020). Being able to efficiently engineer the spatial genome organization or even defined DNA structures in vivo would revolutionize our ability to understand and control cellular systems and address diseases related to chromatin organization defects.
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