Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Abstract

In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5′-TTATT-3′) used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin ( stn ) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. Supplementary Information The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

Design and Evaluation of a Robust CRISPR Kinetic Assay for Hot-Spot Genotyping

“…We also note that ssDNA activation shows stronger trans-cleavage activity than that of dsDNA for both the WT and Mut, which complements the results summarized in Figure and is consistent with the observations of Huyke et al . Note that the Cas12a kinetic parameters measured in this work ( k cat around 0.4 s –1 and K M around 0.3 μM) are comparable to parameters reported by Huyke et al, Rossetti et al, and Lee et al See Table S11 for the kinetic parameters measured in this work.…”

“…We next evaluated reported sequences for ssDNA fluorescent reporters and analyzed three of these in more detail. Lee et al compared 11 reporter sequences to maximize Cas12a trans-cleavage activity. They identified the most efficient sequence (5′-TTATT-CCCCC-3′, hereafter termed 5C) and claimed a 10-fold increase in sensitivity compared to the most commonly used reporter sequence (5′-TTATT-3′, hereafter TTATT).…”

Cas-based bacterial detection: recent advances and perspectives

“…For instance, when employing Cas12a/Cas13a, the presence of cis -cleavage activity can adversely affect amplification efficiency, making it challenging to achieve a one-pot approach, and the cumulative errors introduced through multiple steps are non-negligible. 133,134…”

Sensitive and Direct Analysis of Pseudomonas aeruginosa through Self-Primer-Assisted Chain Extension and CRISPR-Cas12a-Based Color Reaction

“…34 In addition, Han et al proposed an isothermal amplification strategy by employing Cas12a-based primer production for sensitive nucleic acid quantification. 35 The trans-cleavage activity of CRISPR-Cas12a enables highly efficient digestion of ssDNA sequences after recognizing target sequences, 36 making it a promising tool to develop a sensitive and reliable colorimetric method. 37,38 Herein, we propose a novel colorimetric approach for sensitive and reliable P. aeruginosa analysis by integrating selfprimer-based production of ssDNA and CRISPR-Cas12aassisted color development.…”