Fanzor

The Fanzor (Fz) protein is an eukaryotic, RNA-guided DNA endonuclease, which means it is a type of DNA cutting enzyme that uses RNA to target genes of interest. It has been recently discovered and explored in a number of studies.^[1][2][3] In bacteria, RNA-guided DNA endonuclease systems, such as the CRISPR/Cas system, serve as an immune system to prevent infection by cutting viral genetic material.^[4] Currently, CRISPR/Cas9-mediated's DNA cleavage has extensive application in biological research, and wide-reaching medical potential in human gene editing.^[4]

Fanzor belongs to the OMEGA system.^[1][2][4] Evolutionarily, it shares a common ancestor, OMEGA TnpB, with the CRISPR/Cas12 system.^[1][5] Due to the shared ancestry between the OMEGA system and the CRISPR system, the protein structure and DNA cleavage function of Fanzor and Cas12 remain largely conserved.^[1][6] Combined with the widespread presence of Fanzor across the diverse genomes of different eukaryotic species,^[6] this raises the possibility of OMEGA Fanzor being an alternative to CRISPR/Cas system with better efficiency and compatibility in other complex eukaryotic organisms, such as mammals.

Fanzor functions as a potential human genome editor

Due to its eukaryotic origin, the OMEGA Fanzor system may have some advantages over the better studied CRISPR/Cas gene editor in terms of human genome editing applications.^[1] In a CRISPR/Cas9 system, Cas9 proteins are guided by the guide RNA (gRNA) and protospacer adjacent motif (PAM) for DNA cleavage. Interestingly, Fanzor genes in the soil fungus S. punctatus ^[1][5] also contain non-coding sequences called ωRNA. Similar to CRISPR/Cas9, Fanzor protein is shown to cleave DNA in test tubes under the guidance of ωRNA and Target-adjacent motif (TAM).^[1]

 

As shown in the schematic, Cas9 DNA cleavage is instructed by the gRNA and the PAM sequence "NGG"^[7] on the target DNA, where N can be any of the four DNA components (A, G, C or T). Similarly, Fanzor DNA cleavage is instructed by the ωRNA and the TAM sequence "CATA" on the target DNA1. Not an accurate representation of size and structure of the RNAs and proteins. (created using Biorender)

In human cells, the Fanzor protein of Spizellomyces punctatus was successfully tested and shown to cleave DNA effectively.^[1] However, its efficiency is lower compared to the closely related CRISPR/Cas12a system.^[1] By modifying and tweaking the ωRNA and the amino acid sequence, a second version of the S. punctatus Fanzor protein with improved cleavage efficiency - comparable to that of the CRISPR/Cas12a system - was engineered.^[1] This shows that, with better modifications and more research, OMEGA Fanzor has the potential to match the CRISPR system in human genome editing in the future.

Clinical and Biotechnological Significance

Studies conclude that Fanzor has great potential for efficient human genome editing^[1][6] with a higher chance of not getting attacked by the immune system.^[6] For example, Fanzor could be used in personalized cancer treatments where the patient's own T-cells - important cells of the immune system that recognize and fight foreign pathogens - are edited in order to recognize and destroy cancer cells.^[2][8] In the field of regenerative medicine, it offers hope for an application in stem cell therapy to treat many disease of genetic origin like type 1 diabetes or neurodegenerative diseases.^[2]

Furthermore, Fanzor could potentially be used for genome editing in eggs and sperm^[2] for disease prevention and infertility treatment. However, the intervention in such cells' DNA comes with risks and requires strict ethical guidelines.^[9]

One major advantage of Fanzor in comparison to the CRISPR/Cas9 system is its small size. Therefore, it can be delivered with viral vectors, which are modified dead bodies of viruses engineered to safely deliver genetic material, such as adenoviruses.^[4] Adenoviruses are commonly used in medical applications like gene deliveries or vaccines^[10] that do not elicit immune responses within the human body.^[4]

However, researchers caution that further research is necessary to improve the editing efficiency^[1][6] and precision.^[1]

Next to the application in human cells, Fanzor is a prospective tool for specific genome editing in plants, because of the aforementioned advantages of the protein being a small size.^[2] Thereby, the nutrient content, the resistance to diseases and the affordability of crops could be improved.^[11] Moreover, in regard to the current and arising challenges caused by climate change, crops could be adjusted to better endure stress factors such as drought, salinity and increasing temperatures.^[12]

References

1. Saito, Makoto; Xu, Peiyu; Faure, Guilhem; Maguire, Samantha; Kannan, Soumya; Altae-Tran, Han; Vo, Sam; Desimone, AnAn; Macrae, Rhiannon K.; Zhang, Feng (2023-08-01). "Fanzor is a eukaryotic programmable RNA-guided endonuclease". Nature. 620 (7974): 660–668. Bibcode:2023Natur.620..660S. doi:10.1038/s41586-023-06356-2. ISSN 1476-4687. PMC 10432273. PMID 37380027.
2. Awan, Muhammad Jawad Akbar; Awan, Muhammad Raza Ali; Amin, Imran; Mansoor, Shahid (2023). "Fanzor: a compact programmable RNA-guided endonuclease from eukaryotes". Trends in Biotechnology. 41 (11): 1332–1334. doi:10.1016/j.tibtech.2023.08.003. ISSN 0167-7799. PMID 37673694. S2CID 261536553.
3. Bao, Weidong; Jurka, Jerzy (2013-04-01). "Homologues of bacterial TnpB_IS605 are widespread in diverse eukaryotic transposable elements". Mobile DNA. 4 (1): 12. doi:10.1186/1759-8753-4-12. ISSN 1759-8753. PMC 3627910. PMID 23548000.
4. Badon, Isabel Wen; Oh, Yeounsun; Kim, Ho-Joong; Lee, Seung Hwan (2023). "Recent application of CRISPR-Cas12 and OMEGA system for genome editing". Molecular Therapy. 32 (1): 32–43. doi:10.1016/j.ymthe.2023.11.013. ISSN 1525-0016. PMC 10787141. PMID 37952084.
5. Yang, Hui; Patel, Dinshaw J. (2023-11-06). "Fanzors: Striking expansion of RNA-guided endonucleases to eukaryotes". Cell Research. 34 (2): 99–100. doi:10.1038/s41422-023-00894-0. ISSN 1748-7838. PMC 10837191. PMID 37932446. S2CID 265041856.
6. Jiang, Kaiyi; Lim, Justin; Sgrizzi, Samantha; Trinh, Michael; Kayabolen, Alisan; Yutin, Natalya; Bao, Weidong; Kato, Kazuki; Koonin, Eugene V.; Gootenberg, Jonathan S.; Abudayyeh, Omar O. (2023). "Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses". Science Advances. 9 (39): –0171. Bibcode:2023SciA....9K.171J. doi:10.1126/sciadv.adk0171. PMC 10530073. PMID 37756409.
7. Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin (September 2014). "Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease". Nature. 513 (7519): 569–573. Bibcode:2014Natur.513..569A. doi:10.1038/nature13579. PMC 4176945. PMID 25079318.
8. Dimitri, Alexander; Herbst, Friederike; Fraietta, Joseph A. (18 March 2022). "Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing". Molecular Cancer. 21 (1): 78. doi:10.1186/s12943-022-01559-z. PMC 8932053. PMID 35303871.
9. Rubeis, Giovanni; Steger, Florian (2018-07-01). "Risks and benefits of human germline genome editing: An ethical analysis". Asian Bioethics Review. 10 (2): 133–141. doi:10.1007/s41649-018-0056-x. ISSN 1793-9453. PMC 7747319. PMID 33717282.
10. Lee, Cody S.; Bishop, Elliot S.; Zhang, Ruyi; Yu, Xinyi; Farina, Evan M.; Yan, Shujuan; Zhao, Chen; Zeng, Zongyue; Shu, Yi; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; Ho, Sherwin; Athiviraham, Aravind; Lee, Michael J.; Wolf, Jennifer Moriatis; Reid, Russell R.; He, Tong-Chuan (2017). "Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine". Genes & Diseases. 4 (2): 43–63. doi:10.1016/j.gendis.2017.04.001. ISSN 2352-3042. PMC 5609467. PMID 28944281. S2CID 34626858.
11. Pixley, Kevin V.; Falck-Zepeda, Jose B.; Paarlberg, Robert L.; Phillips, Peter W. B.; Slamet-Loedin, Inez H.; Dhugga, Kanwarpal S.; Campos, Hugo; Gutterson, Neal (April 2022). "Genome-edited crops for improved food security of smallholder farmers". Nature Genetics. 54 (4): 364–367. doi:10.1038/s41588-022-01046-7. PMID 35393597. S2CID 248025116.
12. Karavolias, Nicholas G.; Horner, Wilson; Abugu, Modesta N.; Evanega, Sarah N. (7 September 2021). "Application of Gene Editing for Climate Change in Agriculture". Frontiers in Sustainable Food Systems. 5. doi:10.3389/fsufs.2021.685801.