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Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) have exploded into the world of biological research and development due to the rapid adoption of genome engineering tools already developed from a tiny fraction of these microbial adaptive heritable immune systems. Type III-A CRISPR-Cas effector complexes (Csm crRNP) are constructed of multiple Cas proteins formed around a CRISPR RNA (crRNA) containing an 8-nucleotide 5’ tag, from a repeat sequence, together with a (~30-40-nucleotide) spacer sequence, taken from a previous invader. The crRNP is guided by the spacer sequence to bind and cleave complementary (protospacer) RNA via an integral RNase of the crRNP. The work presented here demonstrates that heterologously expressed type III-A systems of Lactococcus lactis, Staphylococcus epidermidis, and Streptococcus thermophilus can be modified and programmed to specifically target diverse endogenous RNAs, causing gene knockdown at the post-transcriptional level. In addition to the specific RNase activity, during crRNP-protospacer RNA binding, a non-specific DNase and cyclic oligoadenylate (cOA) generation become switched on. The cOA binds and activates a trans-acting RNase, Csm6, to degrade encountered RNAs. We found that a short protospacer flanking sequence (PFS) adjacent to the crRNA-protospacer RNA paired region, regulates the crRNP activities. We used heterologous Csm expression systems in combination with target plasmid libraries containing degenerate PFSs, to interrogate a large set of distinct PFSs on crRNP-mediated anti-plasmid immunity. We found that PFSs with potential to base-pair with certain nucleotides of the crRNA 5’ tag result in loss of immune activity. In contrast, PFSs that do not exhibit much or any 5’ tag complementary result in different degrees of function depending upon the precise sequence of the target RNA PFS tested. The results obtained with the heterologous Csm expression systems, that not all PFS sequences function the same in Type III-mediated immunity, were validated in the native Streptococcus thermophilus host. Collectively, our findings reveal that the precise sequence of a target RNA PFS can regulate immune activity. We additionally demonstrate that plasmid interference in S. thermophilus is dependent on activation of the non-specific RNase activity of Csm6, and not the DNase activity of Csm1.