No abstract

CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3′ binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.

CRISPR-Cas adaptive immune systems provide bacteria and archaea with defense16 against their viruses and other mobile genetic elements 1 . CRISPR-Cas immunity 17 involves the formation of ribonucleoprotein complexes that specifically bind and 18 degrade foreign nucleic acids 2,3 . Despite advances in the biotechnological exploitation 19 of select systems, multiple CRISPR-Cas types remain uncharacterized 4 . Here, we 20 investigated a type I-D system from Synechocystis and revealed the Cascade complex, 21 which is required for interference, forms a hybrid ribonucleoprotein complex that is 22 structurally and genetically related to both type I and III systems 5-8 . Surprisingly, the 23 type I-D complex contained multiple functionally-important small subunit proteins 24 encoded from an internal in-frame translation initiation site within the large subunit 25 gene, cas10d. Structural analysis revealed that these small subunits bound the 26 complex similarly to Cas11 small subunits in other type I and III systems, where they 27 are encoded as a separate gene. We show that internal translation of small subunits 28 from within large subunit genes is conserved across diverse type I-D, I-B and I-C 29 systems, which account for ~23% of all sequenced CRISPR-Cas systems 4 . Indeed, we 30 demonstrate that small subunits are expressed from within the cas8c large subunit 31 gene in the Desulfovibrio vulgaris type I-C system. Our work reveals an unexpected 32 aspect of CRISPR-Cas evolution and expansion of the coding potential from within 1 single cas genes. 2 3

Prokaryotes have adaptive defence mechanisms that protect them from mobile genetic elements and viral infection. One defence mechanism is called CRISPR–Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). There are six different types of CRISPR–Cas systems and multiple subtypes that vary in composition and mode of action. Type I and III CRISPR–Cas systems utilise multi-protein complexes, which differ in structure, nucleic acid binding and cleaving preference. The type I-D system is a chimera of type I and III systems. Recently, there has been a burst of research on the type I-D CRISPR–Cas system. Here, we review the mechanism, evolution and biotechnological applications of the type I-D CRISPR–Cas system.