trans Single‐Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction

Abstract

As aC RISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here,w ed escribe an unreported capacity of Cas14a1:R NA can trigger the trans ssDNAc leavage.T his Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNAc leavage,A TCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNAactivation. Using ATCas-RNAv ia af luorophore quencherlabeled ssDNAr eporter (FQ), we were able to detect 1aM pathogenic nucleic acid within 1h,and achieve 100 %accuracy with 25 milk samples.This platform can serve as anew tool for high-efficiency nucleic acid diagnostics.Importantly,this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.

Dual-CRISPR/Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection on Automated Centrifugal Microfluidics

“…In summary, the dual-CRISPR/Cas12a-assisted RT-RAA microfluidic platform has been demonstrated to be a rapid, simple, automated, sample-to-result approach for ultrasensitive and high-specific nucleic acid detection. This platform can be used for CRISPR/Cas12a-assisted RT-RPA or RPA detection and other separate amplification-detection methods based on Cas13a or Cas14 so forth. − Previous reports have proposed the idea of CRISPR/Cas-assisted RPA assay in a microfluidic chip for enabling integrated, sample-to-result, and multiplexed detection. , Our detection platform has realized the requirement of rapid, onsite, automated, and ultrasensitive detection for SARS-CoV-2. Therefore, this straightforward and robust method has great potential in developing a next-generation POC molecular diagnostics technology for the rapid detection of infectious diseases at home or in small clinics.…”

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…According to the collateral cleavage activity of CRISPR-Cas14a, a universal bacterial assay was established by designing extended specific tags on primers, which can simultaneously detect six different bacterial strains with a sensitivity of 1 CFU/mL or 1 aM (Figure 5h) (Song et al, 2021). Wei et al (2021) set up the Cas14a1-based RNA-activated detection platform, which was applied to detect pathogenic bacteria. The method can achieve 1 aM sensitivity.…”

CRISPR/Cas9-based coronal nanostructures for targeted mitochondria single molecule imaging

“…CRISPR/Cas system's precise cutting of nucleic acids makes it an important molecular scissors in the process of identifying and manipulating nucleic acids 24,25 . And, in some reports, the CRISPR/Cas system can respond in a short period of time due to excellent precision and clever controllable strategies [26][27][28] . These properties make it become the candidate for fluorescent signal switches [29][30][31] .…”

“…24,25 And, in some reports, the CRISPR/Cas system can respond in a short period of time due to excellent precision and clever controllable strategies. [26][27][28] These properties make it become a candidate for uorescent signal switches. [29][30][31] But remarkably, when proteins and nucleic acids are the major components of cellular uptake, there are twosided effects.…”