Dokument

Summary:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) is an adaptive immune system in prokaryotes that uses small CRISPR RNA (crRNA) to detect and degrade foreign DNA or RNA. CRISPR-Cas systems consist of a CRISPR array and a set of cas genes. Individual crRNAs are generated from CRISPR array transcripts and are incorporated into CRISPR ribonucleoprotein (crRNP) surveillance complexes that include different Cas proteins. The Type I CRISPR-Cas systems are identified by the presence of the signature protein Cas3 and utilize a crRNP surveillance complex termed Cascade. The Cas protein content of Cascade differs between the CRISPR-Cas subtypes. A minimal variant of the Type I-F subtype was identified in Shewanella putrefaciens CN-32. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. This system contains Cas1, an integrase that mediates spacer acquisition, Cas3, the target DNA nuclease and Cas6f, the endonuclease that generates mature crRNAs. Two additional proteins, Cas1821 and Cas1822, show no apparent sequence similarity to any known Cas protein families. A large subunit protein responsible for target recognition in other subtypes is absent. RNA-Seq analyses showed that the CRISPR array is transcribed and that mature crRNAs are generated. In vivo interference activity was demonstrated for this minimal system using a plasmid conjugation assay. The interference activity was dependent on the recognition of a dinucleotide GG sequence, termed Protospacer Adjacent Motif (PAM). The deletion of cas1821 and cas1822 in S. putrefaciens CN-32 abolished the in vivo interference activity and resulted in the loss of a stable crRNA pool. A minimal Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. Recombinant Cas1821 formed helical filaments upon RNA binding and the analysis of mutant strains demonstrated that DNA interference depended on conserved arginine residues of Cas1822. Cas1822 and Cas3, which contains a conserved Cas2-like N-terminus, are discussed to compensate for the absence of the large and small subunits present in other Cascade assemblies. These results provide insights into the evolution of reduced CRISPR-Cas systems and demonstrate Cascade functionality without a large subunit.

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