Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene

CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution. [Display omitted] •Type I-D Cascade contains Cas11d small subunits expressed from within cas10d•Cas11d is required for specific binding of I-D Cascade to target dsDNA•Alternative Cas11 translation occurs in type I-D, I-B, and I-C systems•Cryo-EM structure of I-D Cascade shows close similarities to type III complexes McBride et al. report the biochemical and structural analysis of the type I-D CRISPR-Cas Cascade complex. They uncover alternative translation of the small subunit from within the large subunit gene. This phenomenon is conserved in type I-D, I-B, and I-C systems, encompassing almost a quarter of all sequenced CRISPR-Cas systems.