Programmable RNA Shredding by the Type III-A CRISPR-Cas System of Streptococcus thermophilus

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA. [Display omitted] •Streptococcus thermophilus type III-A Csm (StCsm) complex targets RNA•Multiple cuts are introduced in the target RNA at 6 nt intervals•Csm3 protein subunits are responsible for endoribonuclease activity of the complex•StCsm complex offers a programmable tool for RNA degradation RNA-protein complexes in CRISPR-Cas systems provide immunity against viruses and plasmids. Type I (Cascade) and II (Cas9) complexes target foreign DNA. Tamulaitis et al. demonstrate that the Csm complex (type III-A) of Streptococcus thermophilus targets RNA and establishes a mechanism for RNA degradation.