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

Abstract

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.

Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets

“… Cas9 Class 2 dsDNA NGG No [ 85 ] Cas12a Class 2 Both (ss/dsDNA) TTTN Yes (ssDNA) [ 5 , 18 , 86 ] Cas12b Class 2 Both (ss/dsDNA) TTN Yes (ssDNA) [ 6 ] Cas13a Class 2 ssRNA – Yes (ssRNA) [ 5 ] Cas13d Class 2 ssRNA – Yes (ssRNA) [ 87 ] Cas14a Class 2 ssDNA – Yes (ssDNA) [ 3 ] …”

Ultrasensitive ImmunoMag-CRISPR Lateral Flow Assay for Point-of-Care Testing of Urinary Biomarkers

“…Additionally, they can pose a risk of contamination by aerosols, which can compromise the accuracy of the sensor. 30 To overcome this problem, alternative isothermal amplification methods have been explored, such as hybridization chain reaction (HCR), 31,32 T7 transcription amplification, 28,29 and multiplication of CRISPR/Cas recognition sites. HCR uses DNA hairpins to generate long, branched DNA structures at room temperature, without the need for specialized equipment or enzymes.…”

“…31,32 RNA amplification by T7 RNA polymerase transcription using DNA templates bound to antigen−antibody assemblies can enhance CRISPR/Cas collateral cleavage activity for protein detection. 28,29 Such trans-cleavage activity is enabled by fluorophore-quencher (FQ) reporters as signaling molecules and a nucleic acid amplification step, such as PCR, 33,34 loopmediated isothermal amplification, 35,36 and RPA, 37 achieve attomolar or greater sensitivity. Overall, this technique offers a highly sensitive and specific way to amplify and detect nucleic acid signals, which has important applications in fields such as medical diagnostics and genetic analysis.…”

“…A major challenge in developing activatable CRISPR/Cas-based detection is to broaden its use beyond nucleic acid targets and to detect other biomarkers, including proteins, organic compounds, and metal ions. − For example, in the case of protein detection, aptamers or antibodies can be used as capture or detection probes and coupled with a CRISPR/Cas-based detection system to achieve high sensitivity and specificity to identify target proteins. − Another challenge is optimizing the amplification step, which is crucial for achieving high sensitivity and specificity . Traditional polymerase chain reaction (PCR) amplification methods are time-consuming and require temperature-controlled devices.…”

“…To overcome this problem, alternative isothermal amplification methods have been explored, such as hybridization chain reaction (HCR), , T7 transcription amplification, , and multiplication of CRISPR/Cas recognition sites. HCR uses DNA hairpins to generate long, branched DNA structures at room temperature, without the need for specialized equipment or enzymes. , RNA amplification by T7 RNA polymerase transcription using DNA templates bound to antigen–antibody assemblies can enhance CRISPR/Cas collateral cleavage activity for protein detection. , Such trans-cleavage activity is enabled by fluorophore-quencher (FQ) reporters as signaling molecules and a nucleic acid amplification step, such as PCR, , loop-mediated isothermal amplification, , and RPA, − to achieve attomolar or greater sensitivity. Overall, this technique offers a highly sensitive and specific way to amplify and detect nucleic acid signals, which has important applications in fields such as medical diagnostics and genetic analysis.…”