Abstract
Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. 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. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.
Highlights
The arms race between viruses on one end and bacteria and archaea on the other end resulted in the evolution of diversified prokaryotic immune systems and viral countermeasures [1,2]
The CRISPR RNAs (crRNAs) are integrated into CRISPR ribonucleoprotein surveillance complexes that are formed by multiple Cas proteins
A minimal Type I-F variant CRISPR–Cas subtype was first described for Photobacterium profundum (PBPRB1995PBPRB1991) [26]
Summary
The arms race between viruses on one end and bacteria and archaea on the other end resulted in the evolution of diversified prokaryotic immune systems and viral countermeasures [1,2]. Upon infection of the host by a virus, a segment of a viral genome, i.e. the protospacer, is integrated as a new spacer into an expanding CRISPR array [12] This stage of the immune response, termed acquisition, relies on the recognition of a short signature sequence, the protospacer adjacent motif (PAM) in the viral DNA sequence [13,14]. The crRNAs are integrated into CRISPR ribonucleoprotein (crRNP) surveillance complexes that are formed by multiple Cas proteins These complexes utilize the crRNA guidance to recognize and degrade foreign genetic material during a recurring infection [6,17,20,21,22]. The recognition of the foreign genetic material is achieved via Watson–Crick base pairing between the crRNA and the unwound target DNA strand or target RNA sequence [23,24,25]
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