Part:BBa_K5237002
SV40 NLS-dSpCas9-SV40 NLS
dSpCas9 is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA (BBa_K5237000) and the dMbCas12a (BBa_K5237001). Transactivation has been shown using this part proving the proper formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.
Contents
Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation,
cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
toolbox based on various DNA-binding proteins to address this issue.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins
include our
finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples that 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 the functionality of our Cas and Basic staples. These
consist of
protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo.
Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of 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 for functional
readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking 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.
Our part collection includes:
| DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly. | ||
| BBa_K5237000 | fgRNA Entry vector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple |
| BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple |
| BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA 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. Can be used to create functionalized staples units |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as 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 to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. 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. 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. It 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 Promotor, mCherry | Readout system for enhancer binding. It 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. It was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence overview
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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) (see FIGURE background Cas9 cas12 panel 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).
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 et al. (2013) established precise targeting of human
endogenous loci by designing the 20 nt spacer sequence accordingly.
Before working with the dSpCas9 the catalytically active version was extensively tested to assess the SpCas9 to be
fit for being part of a Cas staple. The SpCas9 plasmid was provided by our PI.
Wanting to employ the SpCas9 as part of a Cas staple, our goal was to find out how well SpCas9 can stay active
while being transfected together with MbCas12a. Therefore we engineered the dual luciferase assay to allow us to test
two catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla
luciferase gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 3).
For the SpCas9 we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected, but only SpCas9 has a targeting gRNA. When introducing a targeting gRNA for MbCas12a we see no reduction in the highly significant editing of SpCas9, strongly suggesting both Cas proteins to be able to edit simultaneously.
Wanting to test if the different parts of our Cas staples will work together, we tested editing rates of SpCas9
using
fgRNA. Two spacers were tested: FANCF and VEGFA. 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.
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. 5).
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.
Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
potentially giving us another way of Cas stapling. Our goal was to find out how well SpCas9 can stay active while
being
fused to MbCas12a. Therefore we employed the previously engineered dual luciferase assay to allow us for testing
two
catalytically active Cas proteins at once, this time being fused to each other (Fig. 6).
MbCas12a showed highly significant editing rate in the single cut experiment (only MbCas12a has a targeting gRNA).
When introducing a targeting gRNA for SpCas9 no significant change could be detected,
strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
The capability of SpCas9 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this,
the
same target sequences as before were used, namely FANCF and VEGFA in both configurations. Biological
duplicates were done for this assay.
We extensively characterized the catalytically active SpCas9 in several assays asserting functional editing with a
fusion guide RNA (BBa_K5237000), showing high activity while being co-transfected with MbCas9, our second staple
protein's active version, and lastly a functioning fusion to MbCas12a.
The next step was to use the SpCas9 as part of a Cas staple 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. 8, 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. 8, B).
An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.
Continuing the procedure in a similar manner, we focused on inducing proximity between genetic loci using the fusion
dCas next, consisting of dMbCas12a fused to dSpCas9. The same assay was used, with one enhancer plasmid and one reporter
plasmid. Though less distinct than the results for not using a protein fusion, the fusion Cas proteins can be used to
increase expression levels of the reporter firefly luciferase (see figure 13). While using sgRNAs results in similar
relative luciferase activity as for the negative control between 0.1 and 0.2., using a fgRNA with a 20 to 30 nt linker
consistently resulted in activities at 0.25. Fusion guide RNAs without a linker and with a 40 nt linker had on average
about the same activity, but with a higher spread over the biological replicates. Nonetheless this showed the dMbCas12a
fused to the dSpCas9 to be working as a Cas staple. Further tweaking is needed to get better results
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337, 816–821. https://doi.org/10.1126/science.1225829. Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. ACS Chemical Biology, 13, 406–416. https://doi.org/10.1021/acschembio.7b00657. Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nature Biotechnology, 37, 276–282. https://doi.org/10.1038/s41587-018-0011-0. Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox. Biochemistry, 62, 3465–3487. https://doi.org/10.1021/acs.biochem.3c00159. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. (2013). RNA-Guided Human Genome Engineering via Cas9. Science, 339, 823–826. https://doi.org/10.1126/science.1232033. Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., and Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156, 935–949. https://doi.org/10.1016/j.cell.2014.02.001. 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. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., and Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507, 62–67. https://doi.org/10.1038/nature13011.2. Usage and Biology
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). 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. 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).
3. Assembly and part evolution
3.1 SpCas9 can be Co-Transfected wWth other Cas Proteins
3.2 SpCas9 shows editing with fgRNA
Having the sgRNA with single Cas proteins in the same sample resulted in no clear
difference in the editing rates (Fig. 4). 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. Nonetheless we were able to show SpCas9 editing utilizing a
fgRNA.
3.3 SpCas9 can be fused to MbCas12a while maintaining functionality
3.4 SpCas9 fused to MbCas12a shows editing with fgRNA
SpCas9 editing rates were higher overall. At the same time, when targeting VEGFA they resulted in a higher editing
efficiency than FANCF.
4. Results
After all these successful test we were confident to test the Cas staples in action.
4.1 dSpCas9 transactivation as part of a Cas staple
4.2 SpCas9 fused to dMbCas12a form the Cas staple
5. References
| None |