This month’s “In depth” section discusses two new CRISPR gene editing tools from the ocean's depths.
A team from the Institut de Ciències del Mar (ICM-CSIC), in collaboration with the Bellvitge Biomedical Research Institute (IDIBELL), has applied for the patent of two new “gene editing scissors” with unique features that distinguish them from the widely known CRISPR-Cas9 system derived from Streptococcus pyogenes, which is used globally to introduce, remove, or modify genes with precision.
In 2010, the Malaspina Expedition circumnavigated the globe aboard the oceanographic vessel Hespérides to study the impact of global change on the oceans and explore marine biodiversity, particularly that of the deep-sea ecosystems. The expedition resulted in a robust genetic database of microbial life that, even today, hides genetic sequences of interest for biomedicine and biotechnology, such as CRISPR-Cas9 systems that have revolutionized science in this century due to their ability to delete, edit, or introduce new genes into organisms.
The most commonly used "scissors" in laboratories around the world, SpCas9, were discovered in the microorganism Streptococcus pyogenes, a human pathogenic bacterium that thrives in intermediate temperatures. To expand the toolset with gene editing systems that work at different temperatures or target other types of genes, researchers must explore new environments in search of microorganisms that carry them. This is how a team from ICM-CSIC, along with a multidisciplinary consortium, has developed two new tools, for which patents are already in process. These tools were found in a unique and now publicly available genetic database, derived from deep-ocean samples collected during the Malaspina Expedition. Like all CRISPR-Cas9 systems, each tool consists of a Cas9 protein and its associated RNA, which guides the protein to the target DNA location.
Silvia G. Acinas, a microbiologist from the Marine Microbial Ecology group at ICM-CSIC and coordinator of the genetic analysis of the expedition’s samples, led the research to discover these new “gene editing scissors”:
“To expand the variety of CRISPR-Cas9 systems, it is essential to search in different environments where microorganisms might have developed differentiating traits. Our two Cas9s from the deep ocean have distinct biophysical properties that could enable new applications. For instance, one is capable of cutting DNA with great precision at low temperatures,” explains the researcher.
There are numerous scientific articles that describe new CRISPR-Cas9 systems derived from metagenomic databases, which consist of large quantities of genetic information from microbial communities found in environmental samples taken from soils, waters, and even the air. To find these systems, millions are invested in spin-offs and biotech companies like Jennifer Doudna’s Mammoth Biosciences, which was awarded the Nobel Prize in Chemistry in 2020 for the use of SpCas9 in gene editing alongside Emmanuelle Charpentier. However, until now, no one had explored a deep-ocean database as comprehensive as this one. Moreover, not all CRISPR-Cas9 systems that are discovered end up providing useful biotechnological tools.
Two New CRISPR Tools
So far, this database has produced dozens of articles on microbial biodiversity. But now, it has also yielded two novel gene editing tools, DO1 and DO2 (DO for Deep Ocean), both co-invented by Acinas and Julián Cerón Madrigal, a researcher at IDIBELL.
“In the lab, we call them Asterix and Obelix because they are very efficient at cutting DNA”, says Cerón, referring to the famous comic characters who fought the Romans. “Moreover, they work well at colder temperatures than the canonical SpCas9, expanding the range of conditions under which these tools can be used in laboratories,” reveals the expert. Due to the high transfer potential of this project, they have received CERCA GINJOL Patents Fund and BlueNetVal 2025 funding to bring these tools to the market.
“Asterix is smaller than SpCas9 and, therefore, more manageable for biotechnology, while Obelix, which is larger, has a PAM recognition sequence that identifies the target gene, similar to SpCas9. Thanks to this, we can use our Cas9 as an alternative to edit the same DNA regions as SpCas9,” explains Cerón. However, the expert emphasizes that “this is not about replacing SpCas9, which will remain useful, but rather expanding the current gene editing toolbox with new variants like ours”. To explore their full potential, they are now studying their off-target rates (the frequency of unintended DNA cutting) since these new deep-ocean Cas9s appear to be more specific than SpCas9.
The Success of Multidisciplinary Research
For Acinas, the success in developing these two new gene editing scissors is due to the multidisciplinary team behind the work. At ICM-CSIC, researchers Pablo Sánchez and Felipe H. Countinho performed the bioinformatics analysis of the metagenomic database, essentially analyzing millions of genes to identify the most promising ones, which were then tested in the lab to confirm their DNA-cutting activity. “First, we searched for the sequences most similar to the Cas9 protein from the canonical system. From that subset, we selected those that were least similar to SpCas9, the most novel ones, and sent the most promising ones to the lab to confirm whether they cut DNA as predicted,” explains Pablo Sánchez.
Meanwhile, Francesco Colizzi, also from ICM-CSIC, studied the structure and dynamics of the two Cas9 proteins using artificial intelligence tools such as the highly successful AlphaFold, recognized with the Nobel Prize in Chemistry in 2024, which allows 3D computer modeling and studying the interaction between molecules. “These proteins have structural properties similar to the known Cas9, despite having a significantly different sequence”, says Colizzi about his contribution. However, external collaboration was needed to fully characterize the two systems and experimentally confirm which sequences they cut or their specificity in different model organisms.
Acinas explains that this was possible thanks to the National Research Agency 2021 funding to conduct the Proof of Concept of Research and Development Projects, which enabled the creation of the DeepCas expert consortium. She highlights the value of the validations performed on various organisms like plants, fish, microorganisms or in vitro human cells, which were instrumental. The consortium includes the following centers: IDIBELL, the Institute of Molecular and Cellular Biology of Plants (IBMCP), the Andalusian Center for Developmental Biology (CABD), the GENYO center in Granada, and universities from Murcia, Granada, and Copenhagen.
Francis Mojica’s special support
Scientific collaboration has played a key role from the early stages of the project, which also involved one of the “fathers” of CRISPR systems. To decide which sequences to focus on among all those found, the researchers received guidance from Francis Mojica, the person who coined the term CRISPR in 2001 and one of the first to identify this system and associate it with microbial immunity.
“Francis has an immense global vision and knows most of the researchers working on CRISPR-Cas, so it has been wonderful to have his advice and enthusiasm when we discovered these new deep-ocean CRISPR-Cas9 systems”, says Acinas, who celebrates her long-standing friendship with Mojica and his participation in the article they are writing about these findings. Acinas’ career builds on large expeditions with highly diverse teams: “Over the years, I’ve realized that research truly becomes more interesting when it is multidisciplinary”, she concludes.