Characterization of DNA interference by a minimal Type I CRISPR-Cas system
Graduate Students Mini-Symposium
- Datum: 23.07.2018
- Uhrzeit: 13:15
- Vortragender: Hanna Müller Esparza
- MPI Marburg, MPRG Randau
- Ort: MPI for Terrestrial Microbiology
- Raum: Lecture hall
- Gastgeber: IMPRS
- Kontakt: firstname.lastname@example.org
CRISPR-Cas systems represent adaptive immune systems in Archaea and Bacteria. They use ribonucleoprotein complexes containing CRISPR RNAs (crRNAs) to target and degrade viruses in a process termed interference. In Type I CRISPR-Cas systems, the ribonucleoprotein complexes (termed Cascade) are able to discriminate between self and non-self by identifying a Protospacer Adjacent Motif (PAM) near the target. In Type I-E systems, this process is carried out by the large subunit of Cascade and PAM recognition results in the formation of a locked Cascade-DNA complex and subsequent target DNA degradation by Cas3 .
Here, we analyze a minimal Type I-Fv CRISPR-Cas system, identified in Shewanella putrefaciens CN-32. This system lacks a large subunit and encodes two previously uncharacterized Cas proteins, found to be homologues of Cas5f and Cas7f. We expressed the heterologous complex in Escherichia coli BL21-AI and showed Cascade-mediated interference against bacteriophages and plasmids in a sequence-, PAM- and Cas3-dependent manner.
The crystal structure of the Type I-F variant Cascade was solved, revealing that a unique Cas5fv domain is responsible for stringent GG PAM identification . Additionally, we utilized SingleParticle Tracking Photoactivated Localization Microscopy and Biolayer Interferometry to determine the targeting specificities of Cascade.
We propose that this minimized Cascade is an evolutionary response to the appearance of viral anti-CRISPR (Acr) proteins found to block interference by I-F and I-E Cascades. In agreement with this hypothesis, known Acr proteins did not impair the interference activity of this variant complex.
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