Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs

Abstract

SUMMARY Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

“…In such cases, targeting the DNA would counterselect against the transferred CRISPR-Cas as it will kill the host. However, elimination of the resistance element can still be achieved using CRISPR-Cas system that target RNA (12,24,25). Although targeting the RNA will eliminate the resistance conferred by the encoded gene, it will not kill the pathogen and would thus avoid counterselection against the delivering temperate phage.…”

“…CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12)(13)(14)(15)(16)(17).…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…Guide sequences in P. furiosus CRISPRs are generally 37 nucleotides long, and including the 8 nucleotide tag, are 45 nucleotides long. In contrast, the 39-nt species still possesses the 8 nucleotide tag, but lacks 6 nucleotides of the guide sequence at the 3 end [65].…”

“…For a prokaryote that lacks Cmr genes, these can easily be introduced in a single operon along with the engineered crRNA. This opens up potential avenues for microorganism customization and research into gene functions [65].…”

“…Perhaps most importantly, it is also far easier, as the design of crRNAs is simpler than the design of ZFNs or TALENs [65]. Some authors speculate that as the new Cas9/gRNA system improves, it will very rapidly become the nuclease of choice for genetic engineering [116].…”