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Johns Hopkins University
CRISPR-Cas systems are adaptive immune systems found in prokaryotes. Since their discovery, many CRISPR-Cas systems have been characterized biochemically and structurally, and they are divided into two main classes based on their components. Class 1 CRISPR-Cas systems consist of multi-subunit effector complexes (Makarova et al., 2015). Within Class 1, Type III systems are among the most ancient and prevalent CRISPR-Cas complexes found in bacteria and archaea. Consisting of multiple protein subunits and a CRISPR RNA (crRNA), type III effectors specifically bind complementary target RNA, which activates both RNA and DNA cleavage activity to provide defense against invading nucleic acids (Hale et al., 2009; Samai et al., 2015). The subtype III-B complex structure has been solved in many organisms, but little is known about the structural and mechanistic details of type III-B CRISPR-Cas mediated immunity in the system Thermotoga maritima. Previous work in the lab has established the biochemical basis for RNA-activated DNA cleavage by a particular type III-B complex known as the Cas RAMP module (Cmr) in T. maritima, as well as the tolerance of mismatches between the crRNA and the target RNA to elicit this cleavage (Estrella et al., 2016; Johnson et al., 2019). The objective of this thesis was to characterize the structure of the Cmr complex using cryo-EM to validate this biochemical data. The complex was purified by affinity chromatography and analytical gel filtration, the samples were frozen at cryogenic temperature, the complex was imaged, and the resulting images were averaged and processed to obtain a 3D density map of the target unbound complex. We hoped to elucidate the precise catalytic mechanism of target RNA cleavage by the Cmr complex; however, due to low resolution, particle-limited maps and the formation of a sub-stoichiometric subcomplex, we were unable to see the molecular basis for the Cmr complex’s interference activity. Future experiments will focus on optimizing purification protocols and obtaining higher resolution target unbound and target bound structures to investigate specific interactions between the target RNA and key amino acid residues of the complex’s catalytic subunit, Cmr4. This work will not only provide the structural basis for CRISPR-Cas interference in an understudied organism, but it may also give insights that can inform the development of genetic engineering tools.
CRISPR-Cas, structural biology, cryo-EM