A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics

CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

“…Sheng et al developed the Cas-CHDC-powered electrochemical RNA sensing technology (COMET), which comprises a two-stage signal amplification system, through the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor, for high-sensitivity sequential measurements of six non-small-cell lung carcinoma (NSCLC)-related RNAs, including miR-17, miR-155, miR-19b, and miR-210. For the CRISPR/Cas13a system and catalyzed hairpin DNA circuit (Cas-CHDC)-powered RNA detection, all components are thoroughly mixed in a standard reaction vessel and incubated at 37 °C, allowing a dual signal amplification [ 67 ].…”

“…The reusable biosensor platform could selectively and sensitively identify low expression RNA targets in human serum, distinguishing early-stage patients ( n = 20) suffering from NSCLC from healthy subjects ( n = 30) and patients with benign lung disease ( n = 12). The latter demonstrates the ability of the electrochemical CRISPR/CHDC system to be a fast and highly accurate tool for early cancer diagnostics [ 67 ].…”

“…Subsequently, the authors integrated amplification by Cas-CHDC into a reusable electrochemical biosensing technology on a chip to realize rapid and highly sensitive RNA detection on a miniaturized point-of-care testing device [ 67 ]. Thus, miR-17 belongs to the miR-17/92 family of groups whose members of the same family often differ by only 1–2 bases.…”

“…Thus, miR-17 belongs to the miR-17/92 family of groups whose members of the same family often differ by only 1–2 bases. In this regard, the COMET chip was able to distinguish miR-17 from miR-106a (1-base mismatch) and miR-20a from miR-20b (two-base mismatches) [ 67 ]. Moreover, the COMET chip’s ability to detect low abundance target RNAs in complex samples was evaluated by performing a mixture of miR-17 with miR-155 and TTF-1 mRNA at a 1:1000 M ratio (50 fM of miR-17 and 50 pM of non-target RNAs) [ 67 ].…”

“…In this regard, the COMET chip was able to distinguish miR-17 from miR-106a (1-base mismatch) and miR-20a from miR-20b (two-base mismatches) [ 67 ]. Moreover, the COMET chip’s ability to detect low abundance target RNAs in complex samples was evaluated by performing a mixture of miR-17 with miR-155 and TTF-1 mRNA at a 1:1000 M ratio (50 fM of miR-17 and 50 pM of non-target RNAs) [ 67 ]. The authors found that the COMET chip could detect miR-17 in the range of zeptomole and as low as 0.1% of background RNA mixture, which is critical for future clinical application due to the high expression of non-target RNAs in the patient’s bloodstream [ 67 ].…”