The three major types of CRISPR ‐ Cas systems function independently in CRISPR RNA biogenesis in S treptococcus thermophilus

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 S treptococcus thermophilus ( Sth ) strain DGCC 7710, 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 T ype III ‐ A and Cse T ype I – E crRNAs are specifically processed by Cas 6 and Cse 3 ( Cas 6 e ), respectively, and retain an 8‐nucleotide CRISPR repeat sequence tag 5′ of the invader‐targeting sequence. The Cse T ype I – E crRNAs also retain a 21‐nucleotide 3′ repeat tag. The crRNAs from the two Csn T ype 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 Csn 1 ( Cas 9) 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.