Part:BBa_K5237024
TRE-minimal Promoter - Firefly Luciferase
This part contains the tetO binding site (BBa_K5237019), a minimal promoter and a firefly luciferase gene. With a VP64 coming in close proximity to the minimal promoter transcription factors are recruited, initiating expression of firefly luciferase. The described mechanism is utilized in our enhancer hijacking assay for prove of Cas stapling.
Contents
While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D
spatial organization of DNA is well-known to be an important layer of information encoding in
particular in eukaryotes, playing a crucial role in
gene regulation and hence
cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
genomic spatial
architecture are limited, hampering the exploration of
3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
powerful
molecular toolbox for rationally engineering genome 3D architectures in living cells, based on
various DNA-binding proteins.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding
proteins
include our
finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
"half staples" that can be combined by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
successful stapling
and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the
functionality of our Cas and
Basic staples. These
consist of staples dependent on
cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
dynamic stapling in vivo.
We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
target cells, including mammalian cells,
with our new
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom
readout
systems. These include components of our established FRET-based proximity assay system, enabling
users to
confirm
accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a
luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
hijacking events
in mammalian cells.
The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed
exceptional performance as described on our iGEM wiki and can serve as a reference. The other
parts in
the
collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
their
own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome
engineering.
Our part collection includes:
| DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo | ||
| BBa_K5237000 | Fusion Guide RNA Entry Vector MbCas12a-SpCas9 | Entry vector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple Subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
| BBa_K5237002 | Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
| BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity |
| BBa_K5237004 | Staple Subunit: Oct1-DBD | Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237005 | Staple Subunit: TetR | Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237006 | Simple Staple: TetR-Oct1 | Functional staple that can be used to bring two DNA strands in close proximity |
| BBa_K5237007 | Staple Subunit: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237008 | Staple Subunit: rGCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237009 | Mini Staple: bGCN4 | Assembled staple with minimal size that can be further engineered | Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications |
| BBa_K5237010 | Cathepsin B-cleavable Linker: GFLG | Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples |
| BBa_K5237011 | Cathepsin B Expression Cassette | Expression cassette for the overexpression of cathepsin B |
| BBa_K5237012 | Caged NpuN Intein | A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
| BBa_K5237014 | Fusion Guide RNA Processing Casette | Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs |
| BBa_K4643003 | IncP Origin of Transfer | Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery | Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity |
| BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I | Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity |
| BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay |
| BBa_K5237019 | TetR Binding Cassette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 | Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker |
| BBa_K5237021 | NLS-Gal4-VP64 | Trans-activating enhancer, that can be used to simulate enhancer hijacking | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promoter, mCherry | Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker |
| BBa_K5237023 | Oct1 - 5x UAS Binding Casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay |
| BBa_K5237024 | TRE-minimal Promoter- Firefly Luciferase | Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence Overview
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 99
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 99
Illegal BamHI site found at 78
Illegal XhoI site found at 48 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 99
Illegal XbaI site found at 61 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 952
The tetracycline response element, part of the TetR family of regulators (TFRs), is central to the regulation of
antibiotic resistance genes, especially through its role in the Tet operon (TetO). In this system, the TetR
protein
acts
as a repressor by binding to the TetO operator, inhibiting the expression of tetracycline resistance genes. Upon
binding
tetracycline or its analogs, TetR undergoes a conformational change, releasing TetO and allowing the
transcription
of
target genes. This system is widely utilized in molecular biology as a controlled gene expression tool,
particularly
in
inducible gene expression systems in both prokaryotic and eukaryotic cells (Cuthbertson & Nodwell, 2013).
Firefly luciferases are enzymes responsible for the bioluminescence seen in fireflies, catalyzing the oxidation
of
luciferin in the presence of ATP, magnesium ions, and oxygen. This reaction produces light and is highly
efficient,
with
little heat released, making it a popular tool in molecular biology for reporter assays. The gene encoding
firefly
luciferase is widely used in research to monitor gene expression, quantify cellular ATP levels, and study
transcriptional activity due to its sensitivity and ease of detection (Xie et al. (2010))
We utilize the recognition site for plasmid-to-plasmid stapling with our Cas staples. A fgRNA targeting Tre and
Oct1
on
the other plasmid, is the key factor in the Cas staple, bringing them together. When the transactivator,
Gal4-VP64,
binds as well we have transactivation as a readout for functioning staples.
The cloning strategy designed for TetO allows for the easy assembly of repetitive repeats. It follows the
procedure
outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI
and
XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be
linearized
with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated
into
the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility.
This
process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the
oligos.
For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some
limitations regarding sequence depository, the binding cassette is flanked by SalI and XhoI, and the top and
bottom
oligos with the fitting overhangs are annotated.
We were able to show our enhancer plasmid to work great with the Cas staples and the reporter plasmid. For the
whole
assay, an enhancer plasmid and the reporter plasmid were used. The reporter plasmid has firefly luciferase
behind
several repeats of a Cas9 targeted sequence. The enhancer plasmid has the Oct1 being targeted by Cas12a. By
introducing a fgRNA staple (BBa_K5237000) and a Gal4-VP64
(BBa_K5237021), expression of the luciferase is induced
(Fig. 2A).
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. 2B). 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.
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.
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.
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.
Finally, we simulated the behavior of multiple staples at once. Therefore, we expanded our
previously
introduced experimental setup by a second Cas staple.
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). Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor
for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. https://doi.org/10.1038/srep109072. Usage and Biology
3. Assembly and Part Evolution
4. Results
5. In Silico Characterization using DaVinci
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
5.2. Cas staple Forces Do Not Distrub DNA Strand Integrity
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.
than the predicted force typically exerted by a Cas staple on DNA.
than the predicted force typically exerted by a Cas staple on DNA.
than the predicted force typically exerted by a Cas staple on DNA.
than the predicted force typically exerted by a Cas staple on DNA.
5.3. DaVinci Helps to Design Multi-Staple Arrangements
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).
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.
6. References
| None |