There is high demand for a sensitive method for miRNA detection in clinical diagnosis. In this work, we developed a method for miRNA detection based on the surface plasmon-enhanced energy transfer ( SPEET ) between gold nanoparticles ( AuNPs ) and silver nanoclusters ( AgNCs ) , coupled with DNA polymerase and nicking enzyme-assisted isothermal amplification for target recycling. Two DNA probes ( Probe a and Probe b) were assembled onto the surface of AuNPs to form Probe b-Probe a-AuNP conjugates. Probe a consisted three domains:the complementary sequence of miRNA, the specific site of the nicking enzyme, and the self-assembly sequence for AgNCs. The 3′ end of Probe a was modified with thiol as a binding site for AuNPs. The SPEET of AgNCs and AuNPs was inhibited when miRNA was added to produce the dumbbell shaped template by polymerase. The template could promote synthesis of AgNCs, resulting in replacement and subsequently recycling of the target molecule for signal amplification. In comparison with the traditional method of miRNA detection with commercial RT-PCR kits, this method avoided the process of reverse transcription and was easy to perform. In addition, this method with a detection limit of 2. 5×10-11 mol/L was cost-effective, label-free, and highly selective for detecting miRNA, and could be applied to the analysis of miRNA in biological samples.

The CRISPR/Cas is an immune defense system acquired by prokaryotes to resist the invasion of foreign genetic materials during their evolutionary process.In recent years,it has been developed into an efficient tool for genome editing,gene regulation and molecular diagnosis.Its programmable targeting mechanism has opened the door to use this system for genome manipulation and allows for dynamic regu-lation and control of gene expression within its activity range.As one of the most flexible and cost-effec-tive techniques among existing gene modification methods,it has been widely applied in clinical disease treatment,industrial and agricultural production,sustainable dye development,chemical processing and many other fields.With the continuous investigation and exploration of the CRISPR/Cas system,a large number of studies have been reported on the engineering modification and optimization approaches of gRNA,including changing the length of the spacer region,regulating the structure of constant and varia-ble sequence parts,adding extra functional sequences through the end or middle extension,and chemical synthesis modifications,in order to reduce off-target and mutation rates,improve the efficiency of the CRISPR system,and fully stimulate the potential of CRISPR gene manipulation tools in biomedical fields.Based on this,this review will introduce the latest progress in gRNA engineering design strategies and application research of CRISPR/Cas9 and CRISPR/Cas12 systems,analyze and discuss the opportu-nities and challenges in the current gRNA engineering technology,aiming to provide ideas and reference directions for obtaining gRNAs with better performance,thereby effectively improving the ability to probe the human genomes using the CRISPR/Cas system and bringing more possibilities to programmable biology.