Research

The Bailey Lab studies the molecular mechanisms that cells use to interact with and modify DNA and RNA, with a focus on CRISPR systems. Our recent work has provided structural and mechanistic insight into how CRISPR-Cas systems identify and destroy their DNA targets.

The Bailey lab focuses on proteins-nucleic acids interactions. These interactions are critical for how cells work - how they store, use and pass on genetic information - and how they defend against invaders and other threats to maintaining the genome.

Our research has focused on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) – bacterial adaptive immune systems that help protect bacteria from foreign DNA and RNA from viruses and other threats. CRISPR systems use a library of DNA snippets from past invaders, copying them into small CRISPR RNA (crRNA) segments that circulate around the cell. These crRNA guides are checked when foreign DNA shows up, like during a virus attack. Matching DNA gets cut up, preventing infection. Check out our CRISPR Primer for more about these systems.

CRISPR-Cas systems have the potential to be a valuable toolkit. The ability to exploit CRISPR-Cas systems could have applications in protecting domesticated bacteria, such as those used in food and pharmaceutical production, or combating human pathogens and the spread of antibiotic resistance. Additionally, CRISPR-Cas system components are being adapted as tools for gene engineering in a wide variety of organisms, including humans. To fully realize the potential of CRISPR-Cas as a toolkit, we need a more complete understanding of how the systems function. We study the structural and mechanistic aspects of these systems to gain insight into how the CRISPR-Cas systems identify and destroy their targets, with a focus on Type I and Type III systems. We also investigate how the original foreign DNA fragments are acquired and added to the CRISPR library.

The Bailey Lab uses a variety of techniques to gain structural and mechanistic insight into the molecular mechanisms of protein-nucleic acid interactions.

Structural analysis

Though CRISPR and other protein-nucleic interactions can have a major effect at the large-scale, key interactions happen at the atomic and molecular level. To really understand what's going on, we look at the structures of the molecules involved in protein-nucleic acid interactions. Using techniques like x-ray crystallography or cryo-EM, we can determine the structures of the molecules—and how they change when they interact with other components.

Biochemical assays

By isolating the proteins and nucleic acids involved in our systems, we can set up in vitro assays. We can test the conditions needed for the systems to work, such as whether the enzymes need metal ions to function normally. These assays can focus on specific steps, such as binding assays that test how tightly the proteins bind to target DNA. Other assays can test the rate of a step or a series of steps, for example, testing how long it takes to cleave DNA. We can also study the products, such as testing where a particular strand of DNA is cleaved. Often we compare normal and modified proteins or nucleic acid sequences to test out our hypothesis.

The cells in which our systems work are more complicated environments that test tubes, and so we can also test how the systems work in the cells using in vivo assays. For example, we may add plasmids carrying a GFP cassette and target DNA to wild type and mutant cells, and see if the cells can degrade the plasmids – or if the plasmids stay intact, resulting in GFP colonies that glow green under black light.

CASCADE structure

We solved the structure of E. coli’s Cascade bound to a guide crRNA and a single strand of matching DNA and discovered how the complex matches the crRNA and DNA together: not coiling them together like a double stranded DNA helix, but by holding them alongside each other like two sides of a flat ladder. The normally coiled strands are flattened by adding kinks - every sixth base gets a quarter twist, pointing away from the “rungs” of the “ladder,” explaining why most of the bases—the “rungs”—need to match in order for the target to be cut, where as the sixth bases can vary. For more details, check out our post, Viewing a Cascade.

Type III-B System transcription-DEPENDENT DNA CLEAVAGE

We used biochemical methods to characterize how the Type III-B system of Thermotoga maritima functions, and showed that the Cmr complex degrades ssDNA, but only in the presence of RNA that is complementary to the crRNA - a situation that would occur during transcription of the foreign DNA sequence. This helped resolve the ambiguous results from earlier reports about Type III systems. For more details, check out our post, Solving a targeting puzzle: Type III-B DNA cleavage.

Thermotoga maritima Type III system matching requirements

We determined how Thermotoga maritima's type III-B system recognizes its target sequence while distinguishing between native and invader transcripts. We found that the system will allow DNA cleavage even if there are multiple mismatches in the protospacer region, but it is strict in blocking DNA cleavage if even a few bases of the target match the ‘anti-tag,’ which would be found in RNA mis-transcribed from the CRISPR array. For more details, check out our post, What Makes a Match?

E. coli spacer insertion relies on asymmetrical cleavage for spacer orientation  

We showed that during the process of capturing and inserting DNA into the CRISPR array in E. coli, exonucleases trim down the excess DNA on either side of the prospacer asymmetrically, leaving a longer overhang on the side closest to the PAM sequence, and that these asymmetrical overhangs help guide the insertion process to ensure that the spacer is in the correct orientation to make a functional crRNA. For more details, check out our post, CRISPR spacers – asymmetry and orientation.