Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR-Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3. We show that a 4 kb deletion in the SIRV2 genome dramatically reduces infectivity in Sulfolobus islandicus LAL14/1 that carries functional CRISPR-Cas subtypes I-A, I-D and III-B. Subsequent insertion of a single gene from SIRV3, gp02 (AcrID1), which is conserved in the deleted fragment, successfully restored infectivity. We demonstrate that AcrID1 protein inhibits the CRISPR-Cas subtype I-D system by interacting directly with Cas10d protein, which is required for the interference stage. Sequence and structural analysis of AcrID1 show that it belongs to a conserved family of compact, dimeric αβ-sandwich proteins characterized by extreme pH and temperature stability and a tendency to form protein fibres. We identify about 50 homologues of AcrID1 in four archaeal viral families demonstrating the broad distribution of this group of anti-CRISPR proteins.

Prokaryotic CRISPR–Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR–Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3′ and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR–Cas systems.

In the original version of this Article, molecular weight markers in Figs 1c, 2c,d and 4d were displaced during the production process, so that they were not correctly aligned with the corresponding bands. In addition, in Fig. 4c, molecular masses given for three different elution volumes were displaced so that they appeared to the left of the correct positions. These errors have now been corrected.

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