The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs‐CRISPR). Gibbs free energy analysis suggests that the hairpin‐spacer crRNAs (hs‐crRNAs) suppress Cas13a's affinity to off‐target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high‐specificity of hs‐CRISPR/Cas13a system. Compared to ordinary crRNA, hs‐crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs‐crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.

Mass cytometry, also called cytometry by time‐of‐flight (CyTOF), is an emerging powerful proteomic analysis technique that utilizes metal chelated polymer (MCP) as mass tags for interrogating high‐dimensional biomarkers simultaneously on millions of individual cells. However, under the typical polymer‐based mass tag system, the sensitivity and multiplexing detection ability has been highly restricted. Herein, a new structure mass tag based on a nanometal organic framework (NMOF) is reported for multiparameter and sensitive single‐cell biomarker interrogating in CyTOF. A uniform‐sized Zr‐NMOF (33 nm) carrying 105 metal ions is synthesized under modulator/reaction time coregulation, which is monodispersed and colloidally stable in water for over one‐year storage. On functionalization with an antibody, the Zr mass tag exhibits specific molecular recognition properties and minimal cross‐reaction toward nontargeted cells. In addition, the Zr‐mass tag is compatible with MCP mass tags in a multiparameter assay for mouse spleen cells staining, which exploits four additional channels, m/z = 90, 91, 92, 94, for single‐cell immunoassays in CyTOF. Compared to the MCP mass tag, the Zr‐mass tag provides an additional fivefold signal amplification. This work provides the fundamental technical capability for exploiting NMOF‐based mass tags for CyTOF application, which opens up possibility of high‐dimensional single‐cell immune profiling, low abundant antigen detection, and development of new barcoding systems.

Imaging mass cytometry (IMC) is an emerging imaging technology that exploits the multiplexed analysis capabilities of the CyTOF mass cytometer to make spatially resolved measurements for tissue sections. In a comprehensive view of tissue composition and marker distribution, recent developments of IMC require highly sensitive, multiplexed assays. Approaching the sensitivity of the IMC technique, we designed a novel type of biocompatible metal-labeled aptamer nanoprobe (MAP), named 167Er-A10-3.2. The small molecular probe was synthesized by conjugating 167Er-polymeric pentetic acid (167Er-DTPA) with an RNA aptamer A10-3.2. For demonstration, 167Er-A10-3.2 was applied for observing protein spatial distribution on prostatic epithelium cell of paraffin embedded Prostatic adenocarcinoma (PaC) tissue sections by IMC technology. The 167Er-A10-3.2 capitalizes on the ability of the aptamer to specifically bind target cancer cells as well as the small size of 167Er-A10-3.2 can accommodate multiple aptamer binding antigen labeled at high density. The detection signal of 167Er-A10-3.2 probe was 3-fold higher than that of PSMA antibody probe for a targeted cell under lower temperature epitope retrieval (37 °C) of PaC tissue. Furthermore, we successfully demonstrated the simultaneously staining ability of aptamer probes in IMC analysis. The successful imaging acquisition using aptamers probes in IMC technology may offer opportunity for the diagnosis of malignancies in the future.