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.

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

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:

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) (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).

Figure 2: The CRISPR/Cas system (adapted from Pacesa et al. (2024)) 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

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.

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

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.

3.1 SpCas9 can be Co-Transfected wWth other Cas Proteins

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).

Figure 3: 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 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.

3.2 SpCas9 shows editing with fgRNA

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.
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.

Figure 4: 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. 5).

Figure 5: 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.

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.

3.3 SpCas9 can be fused to MbCas12a while maintaining functionality

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).

Figure 6: 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

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.

3.4 SpCas9 fused to MbCas12a shows editing with fgRNA

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.
SpCas9 editing rates were higher overall. At the same time, when targeting VEGFA they resulted in a higher editing efficiency than FANCF.

Figure 7: Editing rates for fusion guide RNAs with fusion Cas proteins. 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 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.
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

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.

Figure 8: 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.

4.2 SpCas9 fused to dMbCas12a form the Cas staple

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

Figure 9: Firefly luciferase trans activation through fusion Cas staple 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). Fusion Cas proteins were paired with sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt

5. References

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.