The three major types of <scp>CRISPR</scp>‐<scp>Cas</scp> systems function independently in <scp>CRISPR RNA</scp> biogenesis in <scp><i>S</i></scp><i>treptococcus thermophilus</i>

TLDR

CRISPR‑Cas systems are small RNA‑based immune mechanisms that defend prokaryotes against viruses and plasmids. The authors aimed to investigate the features and biogenesis of crRNAs in Streptococcus thermophilus DGCC7710, a strain harboring four CRISPR‑Cas systems representing the three major types. They examined crRNA processing in this strain, revealing that each locus’s crRNAs are generated by locus‑specific Cas proteins and associated non‑coding RNAs. The results demonstrate that each CRISPR locus produces distinct crRNA species—Csm III‑A and Cse I‑E crRNAs retain characteristic 5′ and 3′ repeat tags, Csn II‑A crRNAs have unique 5′ truncations and 3′ tags, and Cas9 from one Csn locus specifically generates its crRNAs—showing that multiple CRISPR‑Cas systems can function independently in crRNA biogenesis.

Abstract

CRISPR-Cas systems are small RNA-based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR-Cas systems including representatives from the three major types of CRISPR-Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non-coding RNAs) associated with the respective locus. We find that the Csm Type III-A and Cse Type I-E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8-nucleotide CRISPR repeat sequence tag 5' of the invader-targeting sequence. The Cse Type I-E crRNAs also retain a 21-nucleotide 3' repeat tag. The crRNAs from the two Csn Type II-A systems in Sth consist of a 5'-truncated targeting sequence and a 3' tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR-Cas systems can function independently in crRNA biogenesis within a given organism - an important consideration in engineering coexisting CRISPR-Cas pathways.