Precise Transcript Targeting by CRISPR-Csm Complexes

ABSTRACT Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision, and subcellular compartmentalization. Here, we show that the CRISPR-Csm complex, a multi-protein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immunity pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans -cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13-family enzymes. Using single-vector delivery of the S. thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including shRNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multi-protein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.