Falling in love with bacterial genetics

Marraffini was obsessed with space, astrophysics, and science fiction from a young age while growing up in Argentina. After reading about the advent and promise of recombinant DNA technology in a popular science magazine, his interest shifted toward biology. “During my undergraduate degree in biotechnology in Argentina, I did a lot of DNA manipulation and generated recombinant proteins. This experimental knowledge in molecular biology motivated me to follow a research path,” recollects Marraffini. Because the research opportunities were better in North America compared to Argentina, Marraffini uprooted his young family to pursue a PhD at the University of Chicago.

As part of the PhD curriculum, Marraffini recounts, “I took a class on bacterial pathogenesis and found molecular mechanisms by which bacteria cause diseases fascinating. I found bacteria a great experimental system because many tools were available to mutate and overexpress almost anything. There were also a lot of possibilities to purify proteins of interest using in vitro assays. This is why I fell in love with the bacterial experimental system and ended up joining the laboratory of the course teacher Olaf Schneewind for my PhD.” 

Dissecting CRISPR mechanisms in bacterial immunity

Bacteria are numerous but they are outnumbered by viruses that infect them. CRISPR-Cas is a major immune defense system that evolved in bacteria to fight viruses. Marraffini was interested in how bacteria employ CRISPR mechanisms to interact with and nullify infiltrating DNA and RNA. As a postdoc, Marraffini worked with Eric Sondheimer to experimentally demonstrate for the first time how CRISPR works against conjugative plasmids containing antibiotic resistance. “We showed that CRISPR can prevent the dissemination of antibiotic resistance among bacteria by directly targeting plasmid DNA,” explains Marraffini. This milestone in the CRISPR field was important later for gene editing technology development in mammalian cells. “I collaborated with Feng Zhang at the Massachusetts Institute of Technology. We transplanted a CRISPR-Cas9 system from Streptococcus pyogenes into human hepatocytes and showed that CRISPR cleaves DNA and can be repurposed for gene editing in cells,” shares Marraffini.

Over the years, Marraffini’s group gained mechanistic insights into how CRISPR systems contribute to bacterial immunity. When a phage or a plasmid invades bacteria, the CRISPR system captures a 30- to 40-nucleotides long sequence from the invader DNA called a spacer and incorporates it into the chromosome. This spacer DNA transcribed into the guide RNA gives Cas9—an enzyme that cuts DNA—the target specificity towards invading DNA. This is how bacteria acquire a memory of infection to then fight future infections.

Marraffini also discovered that phage DNA cleavage by Cas9 generates additional DNA fragments, resulting in the acquisition of new spacers for the CRISPR locus. More spacers and guide RNAs against the same-phage DNA are advantageous for bacteria as phages can escape Cas9 cleavage by mutating the target site, offering greater fitness to bacteria. According to Marraffini, “That’s one of our major contributions, showing how spacers acquisition determines infection memory. In addition, we also found that the CRISPR machinery uses free DNA ends, which is a way of diminishing autoimmunity since the bacterial chromosome is circular without a free end.”

Fostering curiosity and boldness

Joshua Modell, Assistant Professor of Molecular Biology and Genetics at Johns Hopkins University School of Medicine, describes his former postdoctoral mentor as “a rare scientist and an intellectual heavyweight who makes the laboratory a stimulating and fun place to do science.” Modell adds, “His ability to interact with and inspire scientists at any career stage, from the greenest summer intern to a long-tenured professor, is what makes him truly special. When I started my postdoc, he explained how much we still had to learn about CRISPR biology and how the work we do could end up in the textbooks. I still try to use that textbook standard with my trainees.”

“My mentors were extremely supportive of my interest in CRISPR despite CRISPR being unknown when I started my academic career,” says Marraffini. He champions the same generosity in his mentorship style, supporting projects his trainees want to pursue. 

“I wanted to investigate a new type of CRISPR that targeted RNA exclusively. No one understood how it worked. While everyone in the laboratory worked on Staphylococcus, I worked on a Listeria strain that naturally carried this RNA-targeting CRISPR system and developed it into a model system,” says Alex Meeske, Assistant Professor in the Department of Microbiology at the University of Washington, who did his postdoctoral training with Marraffini. “He encouraged me to be bold and try new methodologies, even if they were outside his expertise. He taught me to focus, keep my eyes on the prize, and investigate the most significant and testable questions.”

Join us in congratulating Luciano Marraffini, who received the Genetics Society of America Medal at The Allied Genetics Conference 2024 in Metro Washington, DC.

2024 GSA Awards Seminar Series

On September 9, at 1:00 p.m. EDT, Luciano Marraffini will join us to discuss CRISPR-CARF immunity and sacrificing the host for the benefit of the population. Save the date and register here!

Sejal Davla, PhD, is a neuroscientist, science writer, and data scientist with expertise in research in a variety of life sciences. She has more than a decade of experience studying the brain by using cutting-edge methodologies in microscopy, molecular biology, genetics, and biochemistry, and is a motivated storyteller and science communicator.