Researchers at the Zhang Lab and the Abudayyeh-Gootenberg Lab recently discovered the Fanzor protein, a programmable RNA-guided system in eukaryotes that could potentially be more precise and efficient than the CRISPR system in the eukaryotic domain of life. The discovery of Fanzor has had a massive impact on the genetic engineering area of study, as there are now tools to engineer all three domains of life.
CRISPR is a widely reliable gene editing tool to date. It is composed of an RNA-guided endonuclease that leverages extracellular repair mechanisms to edit a gene. The system has been studied extensively and is classified into two classes and six types. It is constantly being refined to reduce off-target effects and increase accuracy, with the goal of creating better gene engineering proteins (i.e., prime editing, base editing, and variants like xCas9). If you want to learn more about that, check out my comprehensive article covering all the basics of CRISPR here:
Fanzor has the potential to be revolutionary in the synthetic biology space. In this article, I will be explaining the discovery of Fanzor, why it’s so important, and how it works. In this article I am simplifying the paper from the Abudyyeh-Gootenberg lab. Note that this is my understanding of the paper, and there can potentially be some mistakes.
Discovery and Intro
RNA-guided nucleases have a significant role in biological processes in both prokaryotes and eukaryotes. The prokaryotic CRISPR-Cas system, for example, provided immunity for bacteria and archaea kingdoms against viruses. However, until this new discovery, our understanding of RNA-guided nucleases was limited to the bacteria and archaea domains:
“People have been saying with such certainty for so long that eukaryotes couldn’t have a similar system” — Ethan Bear
The researchers at the Abuddayeh-Gootenberg lab found out that there was a common ancestor between the CRISPR Cas 12 protein and the Fanzor proteins, bringing up the idea that eukaryotes could also have RNA-guided nucleases.
TnpB proteins are a group of RNA-guided nucleases that are usually encoded by transposons in prokaryotes. A transposon is a type of genetic element that can “jump” between genes to different locations in the genome (mobile genetic element). A protein similar to the TnpB protein named Fanzors was found in different kinds of eukaryotes. The systems in eukaryotes (which include Fanzors) were renamed HERMES, or Horizontally-Transferred Eukaryotic RNA-Guided Mobile Element Systems. (HERMES loci is like the CRISPR loci, and it does not just refer to the nuclease)
Researchers found out many things about Fanzors that convinced them that RNA-guided nucleases exist in eukaryotes, also providing evidence for the evolutionary adaptation of TnpB proteins in eukaryotic cells. For example, Fanzor proteins show that they have nuclear localization signals, a short stretch of amino acids that mediates movement into the nucleus. This is unique to eukaryotic cells, which have a nucleus, a membrane-bound organelle that encases genetic material. Prokaryotic cells, on the other hand, do not have membrane-bound nuclei but rather a highly concentrated part of the cell with circular DNA. Additionally, Fanzor genes indicated the presence of introns, which only eukaryotic genomes carry.
Fanzors show many similarities to Cas proteins. For example, Fanzors have non-coding RNA segments next to endonuclease coding segments. They also contain a similar RuvC domain that doesn’t have collateral cleavage activity and doesn’t indiscriminately cleave gene sequences near it (certain CRISPR systems have this property). Hermes has many similarities to what we know about gene editing. The Abuddayeh-Gootenberg lab’s paper shows how HERMES systems (including Fanzor proteins) can be used to edit eukaryotic cells like human cells. This is game-changing, as it could be 10x better than CRISPR systems as it can better integrate into eukaryotic cell processes.
Prokaryotic and eukaryotic genes are saturated with transposons, or mobile genetic elements. They are often referred to as jumping genes, as they can move seemingly randomly throughout the genome, being excised from one location and inserting themselves into another. Transposases bind to the ends of transposons and move them to new locations. Transposases are encoded by the transposon, making them in a way “self-reliant.”
Transposases of the IS200/605 family, including TnpA and TnpB, are discussed in the paper. TnpA encodes a DDE-class transposase and conducts single-strand transposition. TnpB’s function is not known, but it is unique because it has many similarities to the CRISPR Cas protein. They are also a part of OMEGA systems (obligate mobile element-guided activity), which encode a guide RNA (denoted as ωRNA) next to the nuclease gene. Researchers showed that the ωRNA-TnpB complex is a programmable RNA-guided endonuclease. That can be related to the Cas9-gRNA complex formed in the CRISPR system. The process by which the new complex works is not extremely different. TnpB proteins originating from eukaryotes can be better suited for mammalian gene editing.
Fanzors, more specifically TnpB homologs found in eukaryotes, are found in diverse eukaryotes. These include protists, fungi, algae, amorphae, and large dsDNA viruses. Fanzor proteins are classified into Fanzor 1 proteins, which are associated with eukaryotic transposons, and Fanzor 2 proteins, which are associated with viral ds-DNA genomes. Therefore, they can potentially be used effectively in mammalian cells or even viruses!
“CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome. This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.” — Feng Zhang
Main Research Findings
- Fanzors are widespread in Eukaryotes and Viruses and associate with diverse transposons
- Fanzors associate with HERMES RNA or hRNA — i.e. ApmHNuc is an example
- HERMES nucleases contain a rearranged RuvC domain compared to TnpBs and do not have collateral cleavage activity
- Fanzors can be adapted for mammalian genome editing
HERMES are widespread in eukaryotes and viruses and associate with diverse transposons.
Researchers found RNA-guided nucleases throughout eukaryotic and viral genomes by aligning the RuvC domains of Fanzor1/Fanzor2 proteins with 22497 genomes from the NCBI GenBank. They found 3655 nuclease matches from fungi, algae, protists, and more, as well as many viral families. They found that many genomes contained multiple copies of the nuclease genes, giving evidence that the nucleases are associated with transposons. The researchers created a phylogenetic tree by aligning HERMES with TnpB, showing evolutionary relationships. The tree is split into seven families. Fanzor 1 represents Families 1–4, and Fanzor 2 represents Families 5. The researchers also searched transposon within HERMES sequences and found transposon families like Mariner, Helitron, and Sola (non-random association with the HERMES families).
Fanzors associate with HERMES RNA or hRNA
TnpB is involved in the recognition of transposon ends, cutting or excision, and integration into the new genomic location.
It also processes the transposon RNA (as an OMEGA system) into ωRNA. It then forms a complex with ωRNA that functions like an RNA-guided endonuclease. The researchers found conserved sections of the HERMES loci that could encode non-coding guides that were interestingly longer than TnpB sequences.
Family 5 HERMES proteins were found to be more related to TnpB proteins compared to Family 1–4, making it more likely to find RNA-guided DNA nucleases within it. The researchers looked into the HERMES loci of the Acanthamoeba polyphaga mimivirus encoded in the IS607 transposon. They found that the protein-coding regions (ApmNuc) and the non-coding RNA-coding regions were conserved (repeated). With further study, they found out that the non-coding conserved region could function as a functional guide RNA, and they named it HERMES RNA, or hRNA. It was seen that when hRNA was expressed with ApmNuc in E. coli, the nuclease experienced more stability compared with when it was not expressed with hRNA.
They hypothesized that the ApmHNuc-hRNA ribonucleoprotein (RNP) could target and make double-stranded breaks in DNA. They tested it with synthetic hRNA complementary to a target strand associated with a randomized 7-nucleotide TAM sequence (basically the PAM sequence from the CRISPR system). The cleavage was similar to that of Cas 12 (staggered cuts), with the properties of programmable RuvC domains.
HERMES nucleases contain a rearranged RuvC domain compared to TnpBs and do not have collateral cleavage activity
When comparing HERMES nucleases with the majority of TnpBs, it was found that HERMES nucleases had a proline or glycine residue (not catalytic) in place of a glutamate residue (catalytic) in the RuvC domain. They contained a conserved alternative glutamate about 45 amino acids after, which is very important for the nuclease’s activity and compensates for the substitution mutation. It resembled RuvC domains in mesophilic organisms and was most efficient in the temperature range of 30 degrees to 40 degrees celcius.
The researchers experimented with ApmHNuc and TvoTnpB nucleases (both of which have a rearranged RuvC domain) to see if they had collateral cleavage activity, when the nuclease cleaves surrounding single-strand nucleic acids indiscriminately once it is activated (the target strand is present). For example, Cas12a (which makes double-stranded breaks in dsDNA) after binding to its target DNA can cleave any ssDNA present.
Fanzors can be adapted for mammalian genome editing
As discussed before, eukaryotic endonucleases will need to enter the nuclease to bind to its target DNA, requiring nuclear localization signals. The researchers hypothesized that HERMES nucleases had nuclear localization signals. Using computational methods like AlphaFold, they identified a unique 64-amino acid region at the N-terminus of AmpHNuc. They identified a strong, typical nuclear localization signal (NLS) within this region. With further experiments, they concluded that ApmHNuc has a functional NLS. They also found out that this was reflected in the majority of HERMES nucleases, possibly due to the evolution of TnpB proteins in eukaryotes.
The researchers investigated whether the HERMES nucleases could be used for editing mammalian cells. They modified the ApmHNuc for expression in mammalian cells and designed two guide RNA sequences. They introduced it into HEK293T cells with a plasmid containing the original ApmHNuc protein.
To check if the editing was successful, they created a reporter plasmid with a specific target sequence. If editing occurred, the Gaussia luciferase gene would be expressed resulting in detectable luciferase reactions. There were edits made with the Fanzor protein complex, and this was seen using PCR and next-generation sequencing. There were 2–5 base pair deletions near the 3' end of the target site, which was similar to the patterns seen with programmable nucleases like Cas12.
My Podcast with Michael Trinh — one of the Abuddayeh-Gootenberg lab members!
Gene editing is constantly evolving. This discovery of a gene editing system for eukaryotes can be huge in the gene editing space. The future of the 🌎 is CRISPR and now Fanzor🧬!
Before you go…
You’ve learned about what Fanzors are, how they were discovered, how it compares to CRISPR, and the simplified main research findings (subjective based on my understanding) of the Abudayyeh-Gootenberg lab’s revolutionary paper on Fanzors. You can read it for yourself here: https://www.biorxiv.org/content/10.1101/2023.06.13.544871v1.full
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