2.1 The CRISPR/Cas System as a Gene Editing Tool

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

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.

2.2 Differences between Cas9 and Cas12a

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).

2.3 Dead Cas Proteins and their Application

Figure 3: pre-fgRNA maturation by Cas12a Depicted are the stages of a pre-fgRNA being expressed from the genome, cut by Cas12a into fgRNA molecules forming a RNP with the Cas12a.

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.

3. Assembly and part evolution

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.
Cloning via this strategy resulted in the designed and planned out construct being confirmed by sanger sequencing (figure 4)

Figure 4: Positive cloning of the desired construct confirmed by Sanger sequencing. Two clones were picked and mini prepped after 16 h hours and send to sequencing. Both clones had positive results and clean reads.

4. Results

Due to time constraints we are not able to show data, nevertheless we are actively working on this assay.
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

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.