Abstract

The CRISPR diagnostic (CRISPR-Dx) technology that employs the trans-cleavage activities has shown great potential in diagnostic sensitivity, specificity, convenience, and portability, and has been recognized as the next-generation diagnostic methods. However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.

Highlights

  • The polymerase chain reaction (PCR) amplification and Cas12a trans-cleavage processes are separated in HOLMES and the transfer of amplicons may result in aerosol contamination

  • To precisely calculate the Cas12a trans-cleavage activity, the single-stranded DNA (ssDNA) reporter used was dual labeled with fluorophore and quencher (i.e., FQ-reporter), and the reaction was monitored by a fluorescence reader

  • Non-paired ssDNA FQ-reporters are the substrates for Cas12a trans-cleavage reactions, and the sequences may affect the cleavage efficiencies (Li et al, 2018a)

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Summary

INTRODUCTION

The recently characterized trans-cleavage activities of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas enzymes such as Cas and Cas has undoubtedly sparked the interests in developing CRISPR diagnostic (CRISPR-Dx) systems (Gootenberg et al, 2017, 2018; Chen et al, 2018; Li et al, 2018b; Li Y. et al, 2019). HOLMES first uses the polymerase chain reaction (PCR) technology to amplify the target nucleic acids and detects the amplicons with the Cas12a trans-cleavage activities, illuminating the fluorescent signals. With the use of either tandem crRNAs or remarkably reduced reaction volumes for target nucleic acid detection, diagnostic sensitivities can be greatly improved, and several amplification-free systems have been successfully developed (Fozouni et al, 2021; Liu et al, 2021; Shinoda et al, 2021; Tian et al, 2021; Yue et al, 2021). Dozens of CRISPRDx methods were developed for COVID-19 diagnosis, including the one-pot systems that combine either recombinase polymerase amplification (RPA) with Cas12a (Aman et al, 2020; Ding et al, 2020) or LAMP with Cas12b (Joung et al, 2020), all showing advantages in diagnostic sensitivity, specificity, and convenience (Han et al, 2021). A protocol was provided to simplify the transU definition procedures

RESULTS AND DISCUSSION
MATERIALS AND METHODS
DATA AVAILABILITY STATEMENT
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