Structural basis and rational engineering of a thermophilic Cas13a for robust RNA detection.

Cas13a enzymes are particularly attractive for nucleic acid detection due to their robust collateral activity. Recently, there has been a surge in the discovery and engineering of thermostable Cas13a proteins for nucleic acid detection. The thermostable Cas13a from Thermoclostridium caenicola (TccCas13a) was identified; however, its native target sensitivity and collateral activity are relatively low and require optimization for molecular diagnostic applications. Here, we report comprehensive biochemical characterization and structure-guided engineering of TccCas13a. Our analyses reveal its intrinsic thermostability, nucleotide substrate preferences, and tolerance to structured target RNA substrates. Rational engineering of the spacer-target RNA binding channel improved target engagement and enabled determination of a 3.13 Å cryo-electron microscopy ternary structure. Furthermore, extensive structure-guided engineering of active site-proximal regions yielded a variant with over ∼100-fold enhanced target sensitivity and collateral activity across diverse biological matrices. Together, our work provides mechanistic insights and establishes TccCas13a as a platform for next-generation RNA diagnostics.