Type III-A CRISPR-Cas systems: Structure, Function & Applications towards SARS-CoV-2 diagnosis

Type III-A CRISPR-Cas (Csm) system constitutes a multi-component (Csm1-5, crRNA), multipronged ribonucleoprotein effector complex that confers immunity in bacteria and archaea via foreign-RNA activated and self-RNA inhibited enzymatic activities. Recognition of foreign-RNA triggers indiscriminate single-stranded DNase (ssDNase) and cyclic oligonucleotide (cOA) synthesis activities by the signature subunit Csm1. The newly synthesized cOAs trigger downstream activation of ancillary ribonucleases such as Csm6 adding to the collective immune response. We employed cryo-EM, functional assays and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at various functional states and suggest a possible role of target RNA-induced protein dynamics in activation of the Csm complex.

Additionally, we repurposed the LlCsm complex towards the detection of SARS-CoV-2, as an off-the-shelf COVID-diagnostic, by harnessing both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification allowing virus detection with high sensitivity and at multiple settings. Our detection platform named 'MORIARTY' could reliably detect 2000 copies/μl in amplification-free and 60 copies/μl via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings reliably.