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Microalgae Biodiscovery to Biotechnology: Spotlight on the
I-CreI Meganuclease

Why Study Biodiversity?

Thanks to billions of years of evolution, Earth’s many lifeforms have originated countless biomolecules to survive and thrive in their unique environmental niches. Rather than reinventing the wheel, biotechnology is about harnessing life’s molecular toolkit for human industry and societal benefit. Still, we haven’t really harnessed much of that biodiversity. However, I expect the biotech’s next few decades to include a broader range of bioprospecting efforts for new therapeutics and bioproducts for other major applications like foods, cosmetics, and beyond. 

At Provectus Algae, we often talk about the unique characteristics and biochemical capabilities of algae species. As one remarkably underexplored area of Earth’s biodiversity, algae species undoubtedly contain a deep pool of biotechnologies and biomolecules with significant potential and industrial applications. 

Though we are working to commercialize some of these opportunities using our biomanufacturing toolkit, it’s worth noting just how diverse microalgae biotechnology can be. To help illustrate the potential of the broader algae biotech community, I wanted to share an interesting example of algae’s unique biochemistries: The I-CreI meganuclease and its use as a gene editing technology.

Microalgae Biodiscovery to Biotechnology: I-CreI Meganuclease

The Discovery & Biology of I-CreI

First discovered in a Group 1 Intron within the 23S ribosomal RNA gene of the chloroplast genome of Chlamydomonas reinhardtii, I-CreI is a homing endonuclease belonging to the LAGLIDADG family.

Interestingly, the gene is only expressed when its mRNA is spliced from the primary transcript of the 23S gene. ICre-I, a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves phosphodiester bonds to form a double-strand break (DSB). It’s also an example of a “selfish genetic element” in that it homes in on 23S alleles lacking an I-CreI intron, inserting itself and thus propagating further.

Why Work With Microalgae?

I-CreI Gene Editing Advantages

Given its ability to specifically recognize DNA sequences, form double-stranded breaks, and even insert new DNA, researchers soon realized that I-CreI could form the basis of a gene editing platform independent from CRISPR, alongside others like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

I-CreI’s high sequence specificity, and ability to recognize single base pair differences made it an especially strong target for exploration as gene editing technology. In addition, with a molecular weight of ~38 kDa, I-CreI is much smaller than other gene editing enzymes, which makes it compatible with most engineering and delivery strategies. To put this into context, the CRISPR/Cas9 ribonucleoprotein (RNP) complex reaches ~180 kDa (including the Cas9 protein and its guide RNA). Furthermore, since I-CreI functions as a homodimer and does not require gRNA, researchers only need to deliver a single gene to their targets to start editing the genome. 

While these characteristics of I-CreI make it appealing, its specificity being based on protein-DNA interactions and not RNA-DNA interactions (like with CRISPR-Cas) means that researchers needed to perform derivations to achieve targeting against other sequences to harness it for applications in genetic engineering and gene therapy.

Precision Biosciences ARCUS® Platform

Running with this idea, Jeff Smith, Ph.D., and Derek Jantz, Ph.D., began experimenting with I-CreI while working as postdoctoral fellows at Duke University. Realizing I-CreI’s potential, the pair founded Precision Biosciences in 2006 to further develop and explore its applications.

Precision Biosciences researchers worked to engineer I-CreI to be monomeric by adding a peptide linker between and also used directed evolution to enable engineered I-CreI derivatives to target virtually any sequence. From these developments, Precision Biosciences built its ARCUS® gene editing platform. While the platform’s first uses were on plants, they have since adopted ARCUS to create a clinical pipeline of allogenic CAR-T (ex vivo) and gene therapies (in vivo) to treat various illnesses. 

In 2022, Precision Biosciences reported that interim clinical data from Phase 1/2a study indicated that its lead allogeneic CAR-T candidate had high response rates, making it an early leader for “off-the-shelf” CAR-T therapies. While its allogeneic CAR-T therapies, (PBCAR0191 & PBCAR19B) allogenic CAR-T therapeutics continue to progress through clinical trials, Precision Biosciences has continued to ramp up the use of its I-CreI-derived ARCUS platform as a gene therapy engine, pursuing a number of research programs, including partnerships with Novartis, Lilly, and iEcure.

Mitochondrial Gene Editing

Mitochondrial diseases affect 1 in every ~5,000 people, resulting from mutations in the mitochondrial genome, which includes 37 genes."

One exciting application of I-CreI derivatives (like ARCUS) is its potential for treating mitochondrial diseases. Mitochondrial diseases affect 1 in every ~5,000 people, resulting from mutations in the mitochondrial genome, which includes 37 genes. Today, no cures exist for these diseases. 

Unfortunately, it is historically much more difficult to edit the mitochondrial genome. Mitochondria have a double membrane that significantly limits molecular diffusion. While protein import mechanisms exist, they lack import mechanisms for nucleic acids. This means that CRISPR-based approaches can’t edit the mitochondrial genome. While researchers have explored ZFN and TALEN mitochondrial editing, they often operate as heterodimers, meaning multiple genes must be delivered. Because of their large size, only individual TALEN genes can fit into a single AAV particle. As a result, a double dose of AAVs harboring TALEN genes would need to be delivered, which increases the likelihood of adverse immune reactions in patients. 

On the other hand, I-CreI’s protein-based mechanism, high specificity, small size, and capacity to operate as a single gene makes it particularly well-suited for mitochondrial DNA (mtDNA) editing. A recent study led by Carlos T. Moraes, Ph.D., at the University of Miami, explored the use of ARCUS® I-CreI meganuclease to eliminate disease-causing mtDNA. Researchers found, by targeting ARCUS to the mitochondria (mitoARCUS) and delivering it using AAV9 as a vector, that they could significantly reduce mutant mtDNA in heteroplasmic mice (mice with mitochondrial DNA containing both wild-type and mutant alleles).

While mtDNA gene therapies using I-CreI are still nascent, it seems plausible that this microalgae protein can help make mitochondrial diseases druggable, especially when the conditions cause mitochondrial heteroplasmy. You can check out these posters from Moraes Lab/Precision Bioscience collaboration for additional data.

Exploring Algal Biodiversity

The I-CreI meganuclease represents a great example of how microalgae biodiversity and the novel biomolecules it creates can provide new biotechnological innovations. That said, it’s important to emphasize that researchers discovered I-CreI in Chlamydomonas reinhardtii, probably the most well-studied microalgae species. Put another way, I-CreI may have only been discovered because it resided in model species with significant research attention. This suggests that researcher efforts may turn up other impactful biomolecule products and biotechnologies by more deeply investigating the many thousands of uncharacterized or underexplored algae species. 

Though Provectus Algae does not work on commercializing I-CreI enzymes, the discovery and development of I-CreI shows the impact of building a microalgae biodiscovery library to better understand their diverse biochemistries. Given that only a few species have been fully explored, there’s little doubt that other proteins, enzymes, and biomolecules of industrial value lie in wait. All that is to say, we believe that if the biotech community further studies the massive biodiversity of algae species, we can find valuable opportunities to harness these incredible photosynthesizing biochemists for biotechnologies.

Interested in Exploring Microalgae for Novel Biomolecules?