The Magic of Zinc Finger Nucleases

Meera Singhal
7 min readMay 5, 2021

Have you ever been to Disneyland? Disneyworld? Well, if you have not, it is the happiest place in the world. In Disneyland you will only ever see smiles, it makes it feel truly magical. Do you know what scientists feel is magical though? Gene editing. I mean imagine, you can use different methods of gene editing to physically change your genes and DNA. To me, ZFNs, a form of gene editing are the most magical. Why you may ask? Keep reading to see the true beauty and magic of ZFNs!

Extra info on ZFNs

ZFN’s or zinc finger nucleases is something that we can use to edit the genome by cleaving sections of the DNA. Before I go too deep into ZFNs, I think it would be wise to start with restriction endonucleases. Essentially, restriction endonucleases are an enzyme that can cut DNA at a specific nucleotide sequence. In the e. Coli organism, there is a restriction endonuclease called ecoR1 that targets a specific section of DNA or recognition site. E-coli does this to prevent random cleavages in its DNA that could otherwise be lethal by removing those sites. The zinc finger nucleases can target the specific recognition site. However, the only difference to these two techniques is that we can now customize to a specific nucleotide sequence that the user desires.

Visual diagram of ZFNs

So, what exactly is a zinc finger? So, imagine three ovals. These ovals are in a horizonal line, and they represent proteins. These proteins are held together by a zinc ion (hence the name zinc finger) and they usually work together. Each oval (or protein) is targeting one amino acid. Each ink finger partners with a group of 2–3 other proteins that bind to an enzyme called FOK1. The enzyme (more detail later in this article) is like a pair of scissors. If we have two groups of proteins, we can use these enzymes to cut through the middle through heterodimerizing the two cleavage enzymes. To remove an entire sequence, we need to make a cut.

electroporation

Once we finish this process, we can bring a zinc finger into the cell through transfection which is deliberately introducing nucleic acids or more commonly known as electroporation (which is using a pulse of electricity to open the cell membrane). Once they bind, we can make these cuts and remove the cuts of DNA. By removing this DNA, that DNA is now a deletion mutation. We could also insert a new mutation into this sequence before we inserted this ZFN back into the gene. We would insert the DNA with homologous pairs which would make it an insertion mutation. Essentially, Zinc Finger Nuclease (ZFN) is the binding of a zinc finger protein (DNA binding domain) to a FOK1 enzyme (DNA cleaving domain) to target a specific DNA sequence. Zinc Fingers bind to an amino acid (three bases) and the FOK1 enzyme heterodimerizes to cleave the DNA. A potential application is the use of ZFNs to target the CCR5 gene in HIV patients to prevent the virus from entering a cell.

Gene targeting with homologous repairs

To go deeper into this concept of ZFNs, it is important to know what gene targeting is. What I explained in the last section was an example of gene targeting through ZFNs. Before we jump into the complex world of gene targeting, we should talk about homologous recombination, the base type of gene targeting. Homologous recombination is when genetic information is exchanged through- DNA and RNA; it is only one type of genetic recombination, but it is one of the most crucial. Gene targeting with homologous recombination is a type of technique where scientists can manipulate specific genes to then use another gene editing technique like CRISPR, TALEN, or in our case, ZFNs. Gene targeting through ZFNs grants us the ability to achieve site-specific manipulation, which other techniques do not allow site-specific manipulation as seamlessly as ZFN’s do.

DSB

There are many approaches to gene targeting but double-stranded breaks are the most common in ZFNs. The genome of a cell is continuously damaged, which is inevitable because DNA damage often arises as a result of normal cellular processes. The result is double-strand breaks (DSBs) in the chromosome. A DSB can also be caused by environmental exposure to irradiation, other chemical agents, or ultraviolet lights.

Are you ready for the real magic? All of this information on zinc-finger nucleases, DNA/RNA, DSBs, etc. has been leading up to this portion of the article. That is right, just when you though ZFNs and biology could not get any cooler…

Below, I have listed 5 of the most exciting uses of ZFNs since 2020! Just because CRISPR has the spotlight does not mean that ZFNs are any less cool!

- Inspired by this article, scientists have figured out how zinc finger proteins alongside some proteins promote double-stranded breaks. Though this experiment, scientist tried to make ZFN proteins (one of the largest protein families), cooperate with two other proteins. The ZFN protein E4F1 cooperated with the PARP-1 and the BRG1 protein. After culturing these proteins, scientists saw that their experiment was a success, it promoted DNA double-strand break repairs!

- Inspired by this article, scientists are using ZFNs along with other technology to possibly detect and cure cervical cancer! Through this fascinating experiment, scientists combined Cisplatin and Trichostatin A with ZFNs to create an antitumor efficiency in cervical cancer cells. This solution was inspired by the struggle of different types of persistent and high-risk infections from different genes (HR-HPV) which is the leading cause of cervical cancer. This gene expresses oncoproteins E6 and E7, the proteins that are almost guaranteed to cause cervical cancer. The goal of this project was to use develop an anti-cervical cancer drug with ZFNs. They are still testing this drug out, but it shows great promise!

- This article inspired scientists to use ZFNs to target the cholera toxin A (ctxA) gene. The purpose of this experiment is to use ZFN and ZFN technology to prevent (and disrupt the process) of the cholera toxin gene from inhibiting CT toxin production in vitro. The approach that they are using biologically engineering a ZFN that was designed to target the catalytic site of the cholera toxin A gene. The efficiency of this method was evaluated through a process called colony counting. ZFN might have off-target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism.

- Inspired by this article, scientists have discovered a new use of ZFNS, they are using Zinc-finger activators to use protein delivery to penetrate HIV cells! This technology, if it works, has so much potential to cure or at least relieve the effects of HIV! HIV has been around for hundreds of years, a den yet scientists are no where near a cure. There has not been much success in relieving some of the side effects either, but in this paper, scientists propose a new idea. They are proposing an alternative approach for stimulating latent HIV-1 expressions through direct protein delivery of a cell-penetrating ZFA (zinc finger activator).

- This article inspired scientists to use Codon-swapping of zinc-finger nucleases to confer expressions in primary cells and in vivo from a single lentiviral factor. The inspiration and drive of this project stems from the delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. They used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. They reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines.

TLDR

- What is ZFN? ZFNs are a form of gene targeting. Check out my YouTube Video for a more visual explanation.

- Gene Targeting. Through homologous recombination, gene targeting is possible.

- DSB. DSB or double-stranded breaks are a common form of ZFNs.

  • Case Studies/ examples! 5 of the most popular uses of ZFNs since 2020!

Hello! My name is Meera Singhal, and I am a 13-year-old currently fascinated by the field of biotechnology, specifically stem cells and gene editing. I’ve written articles about biotechnology, mindset tips, and a whole variety of up-and-coming topics. Interested? Check out my medium, LinkedIn, YouTube, or TKS Life Portfolio for more content! Curious to see more about me? Consider subscribing to my Newsletter! Thank you so much!

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Meera Singhal

Biotech Enthusiast at TKS | Stem Cells | Gene Editing