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Julie’s Biggest Takeaways
CRISPR is a bacterial immune system that identifies and destroys specific nucleotide sequences. These sequences are most commonly associated with foreign DNA from bacteriophage or plasmids.
Bacterial acquisition of new CRISPR spacer sequences is fairly inefficient, and often a bacterium dies before acquiring and fending off a new phage infection. Only about 1 in a million cells emerge from a phage infection with a new spacer sequence, likely driven defective phages that act as a vaccine of sorts to provide spacer sequence material.
40% of bacteria and 85-90% of archaea have had some sort of CRISPR system detected in their genomic sequences.
Most bacteria have Type I CRISPR system. This system includes different proteins that serve unique functions: one holds onto CRISPR RNA, one helps identify complementary sequences, and one cleaves the actual nucleotide sequence. The Type II CRISPR system has a single protein, Cas9, which performs all of these functions by itself. Because of its simplicity, this Type II CRISPR system has become widespread as a DNA manipulation tool.
What are the inputs to CRISPR? How do bacterial cells turn CRISPR genes on and off? Do CRISPR systems serve any other regulatory functions? There are still a number of questions that need to be answered to understand the biological role of CRISPR systems.
“Since we study these interactions from the phage perspective, I like to describe CRISPR as a shark in the water with its mouth open. The phage has to jump in the water and quickly produce inhibitors or do whatever it can to stay afloat before it gets chewed up. In my shark analogy, this is a bacterium that of course already has a spacer that’s going to target the phage, so there’s blood in the water and the shark is hungry and ready to eat.”
“There’s still a lot to discover. Most of what we know is still drawn from a few model systems: E. coli, Bacillus subtilis. As you broaden your horizons from one model to another, you discover a lot of new information. Our goal is to go beyond the traditional models.”
“In nature, what’s exciting is that CRISPR-Cas systems have independently arisen multiple times. The Cas9 that cuts in Type II systems and the Cas3 that cuts in Type I systems: they are not homologous genes. They don’t share a close common ancestor; these enzymes have emerged independently. We now have 6 distinct types of CRISPR systems that have likely come up independently and many of the proteins that comprise them have come up independently over the course of evolution.”
“If you ignore CRISPR as a tool for a second, think about all the types and flavors of CRISPR systems we have! Bacteria have not just arrived at one system and held onto it. There’s been a lot of divergence but there’s also been a lot of innovation and other proteins from other DNA and RNA bindings sytems have been brought into CRISPR-Cas function.”
“It’s a high-stakes game: if you don’t protect yourself from phage, it’s a dead end.”
“The experimental system is really the most important consideration when going into a new area, where you can innovate, move quickly, and test ideas.”
Links for This Episode
- Take the MTM listener survey (~3 min.)
- Joe Bondy-Denomy UCSF Lab Website
- Rauch BJ. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell 2017.
- Borges AL. Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity. Cell 2018.
- Mendoza SD. A Nucleus-Like Compartment Shields Bacteriophage DNA from CRISPR-Cas and Restriction Nucleases. bioRxiv 2018.
- UCSF Sandler Fellows Program
- HOM Tidbit: Coming of Phage Celebrating the Fiftieth Anniversary of the First Phage Course
History of Micobiology Tidbit
I want to focus on a piece of advice Joe gave at the end of our conversation: consider your experimental system. This was something that scientists should absolutely do individually as they consider their individual careers, but it’s also a consideration to be done by scientific communities to consolidate model organisms and decide what important questions to focus on. One of the first scientific groups to deliberately outline an experimental system was The Phage Group. This is a group consisting of many famous scientists whose names you likely recognize - Emory Ellis, Salvador Luria, Alfred Hershey, Renato Dulbecco, Matthew Meselson, and a dozen others. This scientific group was brought together by Max Delbruck, a physicist-turned-biologist seeking a simple experimental system to study the fundamental laws of life. Arriving at Caltech in 1938, some biographers suggest that Delbruck was already convinced that viruses, and particularly bacteriophage, would be a superior system for unlocking the secrets of the gene.
The phage group was formed as an informal cohort of scientists working at different universities. Many of the group members had already been working with bacteria and bacteriophage to discover the nature of genetic regulation and the biochemical basis of metabolism. But how could experiments made in one system be validated by use of another? How would results be proven to be robust and reproducible if each group studied its own phage?
These issues were partially solved by formation of the phage group but more formally addressed when Delbruck composed the Phage Treaty in 1944. These guidelines suggested all scientists in the phage group should focus on the 7 “T” series phages and use E. coli strain B, which would allow experiments to be easily replicated in other phage labs. Delbruck further suggested standardized growth conditions and media so results would be comparable. The phage group also set up a course on bacteriophage at Cold Spring Harbor Laboratory on Long Island, which Delbruck himself taught for a number of years.
The phage treaty didn’t last very long. As phage biology increased in popularity, thanks in part to the course, and new scientists wanted to dive into additional topics of phage growth and basic biology, these scientists began to adopt other systems more suited for their questions. The treaty was broken and study of the lambda phages in E. coli K-12 took off, becoming the more dominant system of the 1950s and 1960s. Studies in the lambda/K-12 system led to discovery of the operon, genetic repression, and the genetic code itself, so you could say that breaking the treaty was overall a good decision.
I did an extensive search but couldn’t find any written record of the Phage Treaty itself, rather I found a number of writings about the phage treaty. Was it ever written down? Send it my way if you know where I can find it by tweeting at us @ASMicrobiology or leaving a comment on facebook.com/asmfan.
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