Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

CRISPR-based biosensing technology has been emerging as a revolutionary diagnostic tool for many disease-related biomarkers. In particular, RspCas13d, a newly identified RNA-guided Cas13d ribonuclease derived from Ruminococcus sp., has shown great promise for accurate and sensitive detection of RNA due to its RNA sequence-specific recognition and robust collateral trans-cleavage activity. However, its diagnostic utility is limited to detecting nucleic-acid-related biomarkers. To address this limitation, herein we present a proof-of-concept demonstration of a target-responsive CRISPR-Cas13d sensing system for protein biomarkers. This system was rationally designed by integrating a dual-aptamer-based transcription amplification strategy with CRISPR-Cas13d (DATAS-Cas13d), in which the protein binding initiates in-vitro RNA transcription followed by the activation of RspCas13d. Using a short fluorescent ssRNA as the signal reporter and cardiac troponin I (cTnI) as the model analyte, the DATAS-Cas13d system showed a wide linear range, low detection limit, and high specificity for the detection of cTnI in buffer and human serum. Thanks to the facile integration of various bioreceptors into the DATAS-Cas13d system, the method could be adapted to detecting a broad range of clinically relevant protein biomarkers, and thus broaden the medical applications of Cas13d-based diagnostics.