Staple Subunit: dMbCas12a-Nucleoplasmin NLS

dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.

Figure 1: How our part collection can be used to engineer new staples

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:

1. Sequence Overview

2. Usage and Biology

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) (Figure 2 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).

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

3. Assembly and Part Evolution

3.1 Qualitatively Assessing Gene Editing of Cas12a Orthologs

To select a suitable Cas12a ortholog for constructing the Cas staples, three different orthologs were ordered from Addgene: AsCas12a (#69982), LbCas12a (#69988), and MbCas12a (#115142).

We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and FANCF. For comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.

Figure 3: Preliminary T7 Endonuclease I Testing of Cas12a Orthologs. T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 functions as a benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and FANCF were targeted. Editing is indicated by an extra band compared to the negative control.

3.2 Quantitative Comparison Between AsCas12a and MbCas12a

Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish between the better editing ortholog.
To accurately quantify the editing efficiency, we conducted a dual luciferase assay. This assay measures the luminescence of firefly luciferase, which decreases proportionally to the editing efficiency at the target site. To account for variations in cell count and transfection efficiency, the luminescence is normalized to Renilla luciferase, which acts as an internal control (Fig. 4). The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency compared to AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.

Figure 4: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Assay. Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group between the same Cas proteins are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

3.3 Multiplex Gene Editing Using MbCas12a and SpCas9

To employ the MbCas12a as part of a Cas staple, we sought to determine MbCas12a's activity while being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing 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. 5).

Figure 5: Testing for Simultaneous Editing with Double Cut Luciferase Assay. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed contains MbCas12a and SpCas9. Cas12a are the firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected, but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we did not observe reduction in the highly significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.

3.4 Fusion Guide RNA Enabled Editing with MbCas12a

To further confirm that MbCas12a can be combined with fgRNAs in a functional Cas staple, editing rates were tested using a fusion guide RNA (fgRNA, BBa_K5237000) targeting two different loci: 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.
Having the sgRNA with single Cas proteins in the same sample resulted in no clear difference in the editing rates (Fig. 6). 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 MbCas12a editing utilizing a fgRNA.

Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a. In A and B the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. A and B display both orientations of the two spacers for VEGFA and FANCF.

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. 7).

Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) ^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5.

3.5 Establishing Functional Fusion Cas Proteins with MbCas12a and SpCas9

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 MbCas12a can stay active while being fused to SpCas9. 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. 8).

Figure 8: Double Cut Dual Luciferase Assay Testing Fusion Cas Simultaneous Editing. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas contains MbCas12a and SpCas9. MbCas12a are the firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a, strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.

3.6 The Combination of Fusion Guide RNAs and Fusion Cas Proteins

The gene editing efficiency of MbCas12a 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. We included biological duplicates in this assay.
MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they resulted in a higher editing efficiency than FANCF.

Figure 9: Editing Rates for Fusion Guide RNAs with Fusion Cas Proteins. In A and B the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked as individual dots.

4. Results

We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out of three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion to SpCas9.
After all these successful test we were confident to test the Cas staples in action.

4.1 dMbCas12a as Part of a Cas Staple Establishes DNA-DNA Proximity

The next step was to use the MbCas12a as part of a Cas staple to bring two DNA loci together, and thereby induce proximity between two separate functional elements. Mimicking the phenomenon of enhancer hijacking, in which an enhancer activates an otherwise distant promoter, we constructed artificial enhancer and reporter plasmids. The reporter plasmid encodes a firefly luciferase downstream of 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. 10A). Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2. Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.

Figure 10: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

5. References

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.

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.

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.