CRISPR/Cas12a collateral cleavage activity for an ultrasensitive assay of RNase H

Abstract

We herein describe an ultrasensitive RNase H assay by utilizing CRISPR/Cas12a collateral cleavage activity. In this technique, a chimeric duplex probe consisting of activator DNA and its complementary blocker RNA...

An ultrasensitive label-free RNase H assay based on in vitro transcription of fluorogenic light-up aptamer

“…5, the samples containing the two cell lysates showed substantial uorescence enhancements, indicating that both cell lysates possess high RNase H activities. Notably, the relative RNase H activities in the two cell lysates fully agreed with those from previous reports, 15,34 verifying the reliability of the designed strategy. To verify that the uorescence enhancements were due to the activity of RNase H in the cell lysate and not to any nonspecic enzymes, we conducted the same experiment using HP containing only DNA stems (DNA HP).…”

“…5, the samples containing the two cell lysates showed substantial uorescence enhancements, indicating that both cell lysates possess high RNase H activities. Notably, the relative RNase H activities in the two cell lysates fully agreed with those from previous reports, 15,34 verifying the reliability of the designed strategy. To verify that…”

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3′–5′ Exonuclease Assay

“…In contrast, when a complex with ssDNA is formed, DNA cleavage can occur even without the PAM site [26]. After the Cas12a/gRNA/DNA complex is formed, it randomly cleaves nearby ssDNAs via collateral cleavage [24,26,27]. Similarly, Cas13a, after performing cis-cleavage on a ss RNA, undergoes a transformation into a nonspecific endonuclease with the ability to cleave nearby ss RNA sequences without discrimination [28].…”

“…For instance, various nucleic acid detection methods have been developed, including the one-hour low-cost multipurpose highly efficient system (HOLMES) [29] and Specific High sensitivity Enzymatic Reporter unlocking (SHERLOCK) [28]. Furthermore, recently, advancements have been made to detect not only nucleic acids but also the activity of various enzymes and heavy metals using CRISPR-associated proteins [27,[30][31][32]. However, despite these numerous advancements, progress in the rapid detection of non-nucleic acid substances such as enzyme activity lags behind that of nucleic acid detection using the CRISPRs/Cas system [33].…”