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Schematic of the smLiveFISH system using multiplexed guides against a target RNA to achieve single-molecule resolution. Upon transfection with Csm and CRISPR array plasmids, cells produce Csm1, Csm2, dCsm3-2xGFP, Csm4, Csm5 and Cas6 proteins along with the pre-crRNA. Cas6 processes the pre-crRNA into individual crRNAs that assemble with Csm proteins into RNPs. RNPs, each with their own crRNA spacer, bind target RNA molecules simultaneously through base-pair complementarity, allowing RNA detection at single-molecule resolution.

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