Part:BBa_K5237014
fgRNA processing casette
Incorporating Cas12a into our Cas staple design allows us to utilize the ability of processing its own pre-crRNA by recognizing the hairpin structures of the scaffolds. The cutting of the pre-crRNA into crRNA happens upstream of the scaffold, suggesting the Cas12a to be able to process a CRISPR-array of fgRNA, while maintaining functionality.
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 276
Illegal XhoI site found at 305 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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) (see figure 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 RNA (sgRNA)s, 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.
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 to the Cas9 gRNA and positioned on the 5' side of the crRNA
(see figure 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 5nt long (Paul and Montoya, 2020).
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 RNA (fgRNA)s. 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.
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.
Due to time constraints we are not able to show data, nevertheless we are actively working on this assay. 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. 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. Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., and Kim, Y. (2017). Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature Communications 8. https://doi.org/10.1038/s41467-017-01650-w. 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. Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal 43, 8–17. https://doi.org/10.1016/j.bj.2019.10.005. 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. Wu, T., Cao, Y., Liu, Q., Wu, X., Shang, Y., Piao, J., Li, Y., Dong, Y., Liu, D., Wang, H., Liu, J., & Ding, B. (2022). Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System. Journal of the American Chemical Society, 144(14), 6575-6582. https://doi.org/10.1021/jacs.2c01760. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163, 759–771. https://doi.org/10.1016/j.cell.2015.09.038.2. Usage and Biology
2.1 The CRISPR/Cas System as a Gene Editing Tool
2.2 Differences between Cas9 and Cas12a
2.3 Dead Cas Proteins and their Application
3. Assembly and part evolution
Cloning via this strategy resulted in the designed and planned out construct being confirmed by sanger sequencing (figure 4)
4. Results
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 up 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. References