Dokument

Summary:
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute the only known adaptive immune system present in Archaea and Bacteria. This system targets and degrades foreign genetic material through ribonucleoprotein effector complexes carrying CRISPR RNAs (crRNAs), in a process termed interference. CRISPR-Cas systems are classified into 6 different types, with Type I systems being the most widespread in nature. They harbour the Cas3 helicase/nuclease as signature protein, involved in target degradation, and can be divided into 7 subtypes (A-F, U). They elicit interference by ribonucleoprotein complexes (termed CRISPR associated complexes for anti-viral defence, Cascades), formed of Cas proteins and a single crRNA. Cascades are able to discriminate between self and non-self substrates by identifying a short Protospacer Adjacent Motif (PAM) next to the crRNA target (termed protospacer). In Type I-F systems, PAM recognition is carried out by Cas8f (or large subunit). In the present work, we analysed a smaller variant of a Type I-F CRISPR-Cas system, identified in Shewanella putrefaciens CN-32. This system lacks a large subunit and contains two previously uncharacterised Cas proteins, functionally identified as Cas5 and Cas7 homologues. We expressed the complex in Escherichia coli BL21-AI and demonstrated its activity against bacteriophages and plasmids in a sequence-, PAM- and Cas3-dependent manner. This heterologous activity indicates that interference can be carried out without the need of a large subunit or additional proteins from S. putrefaciens. Furthermore, we were able to identify a unique alpha helical domain in Cas5fv responsible for stringent GG PAM identification from the major groove side, in addition to Cas7fv-mediated non-target strand stabilisation. We also determined the binding affinities of the complex through BioLayer Interferometry (BLI), gaining insights into its target requirements. Moreover, we studied the dynamics of the minimal system through Single-Particle Tracking Photo-activated Localization Microscopy (sptPALM), revealing an unspecific DNA-binding capacity of the alpha helical domain of Cas5fv, as well as RNA interactions of Cas5fv and Cas6f. For Cascades, we determined the binding times to genomic targets, which were directly proportional to the complementarity between crRNA and DNA. Fully complementary targets elicited a binding duration of around 15 seconds. We propose that this minimised Cascade version is an evolutionary response to the appearance of viral Anti-CRISPR (Acr) proteins, small proteins able to block CRISPR-Cas interference. The effect of a broad-spectrum Acr protein, AcrF9, on the minimal complex was tested. Neither a reduction of interference activity nor physical interactions were detected. This supports the hypothesis of complex reduction as a response to Acr pressure.

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