CRISPR-based biosensing technology has been emerging as a revolutionary diagnostic tool for many diseaserelated 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-acidrelated 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.

Orthogonal therapy that combines CRISPR-based gene editing and prodrug-based chemotherapy is a promising approach to combat multidrug-resistant cancer. However, its potency to precisely regulate different therapeutic modalities in vivo is...

The cGAS‐STING (cyclic GMP‐AMP synthase/stimulator of interferon genes) pathway is emerging as a promising target for cancer immunotherapy. However, developing specific and effective strategies for activating the cGAS‐STING pathway in tumors is still challenging. Here, a microRNA‐21 (miR‐21)‐responsive nucleic acid system, as a STING signal amplifier, is designed based on the branched catalytic hairpin assembly (bCHA). The effects of three types of dsDNA structures (linear dsDNA, Y scaffold dsDNA, and dsDNA dendrimer) on cGAS‐STING activation are systematically studied. This study demonstrates that dsDNA dendrimer can induce the most effective liquid–liquid phase separation in a miR‐21‐dependent manner, allowing the controllable activation of cGAS‐STING in both cancer cells and dendritic cells. Given the programmable nature of nucleic acid structure, this study will enable a readily accessible platform for integrating with immunotherapeutic strategies while opening new avenues for controllable, tumor‐specific activation of STING agonists.

CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg 2 + to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn 2 + and Ca 2 + could replace Mg 2 + as cofactors of LbuCas13a. In contrast, Ni 2 + , Zn 2 + , Cu 2 + , or Fe 2 + inhibits the cis-and trans-cleavage activity, while Pb 2 + does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg 2 + and Mn 2 + can further enhance the transcleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.