MeCas12a, a Highly Sensitive and Specific System for COVID‐19 Detection

Abstract

Cas12a-based systems, which detect specific nucleic acids via collateral cleavage of reporter DNA, display huge potentials for rapid diagnosis of infectious diseases. Here, the Manganese-enhanced Cas12a (MeCas12a) system is described, where manganese is used to increase the detection sensitivity up to 13-fold, enabling the detection of target RNAs as low as five copies. MeCas12a is also highly specific, and is able to distinguish between single nucleotide polymorphisms (SNPs) differing by a single nucleotide. MeCas12a can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples and distinguish between SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in simulated samples, thus offering an attractive alternative to other methods for the diagnosis of infectious diseases including COVID-19 and MERS.

CRISPR-Based COVID-19 Testing: Toward Next-Generation Point-of-Care Diagnostics

“…They studied the crystal structure of Cas12a to form a hypothesis regarding crRNA extension and its influence on collateral cleavage activity. Interestingly, this study produced results that contradicted those of Ma et al, 2020. The authors found out Mn 2+ inhibited the LbCas12a activity, in contrast to Mg 2+ , which increased the activity. This contradiction should be further studied with the same controls to determine the reason behind the two different results.…”

“…They demonstrated that using Mn 2+ increased the efficiency and, most importantly, the specificity. Thus, they termed this technique manganeseenhanced Cas12a (MeCas12a) (Ma et al, 2020). It has been found that Mn 2+ helps to stabilize the most stable bond between CRISPR and target DNA (Sundaresan et al, 2017).…”

“…It appears that the performance of the CRISPR protein can vary depending on which organism it is isolated from, (Gootenberg et al, 2018;Ali et al, 2020;Nguyen et al, 2020). For example, addition of Mn 2+ does not improve the sensitivity of all Cas12a proteins, because of the differing function depending on which organism the Cas12a is isolated from (Ma et al, 2020). There have also been contradictions between some studies.…”

“…There have also been contradictions between some studies. One study mentioned that Mn 2+ improved the sensitivity of LbCas12a, whereas another study stated that it inhibited the activity of LbCas12a (Ma et al, 2020;Nguyen et al, 2020). It is important to choose the right organism, the right protein, and the right kind of modification.…”

A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

“…To elevate the detection power, people have been mainly focusing on selecting better effector components (e.g., Cas nuclease, gRNA, reporter, and polymerase), adjusting the stoichiometry of different constituents (e.g., Cas protein:gRNA ratio) 19, 25 or modifying gRNA structure to augment trans-cleavage activity of Cas protein 18 . In addition to manipulating detection components, changing the reaction environment by either optimizing buffer condition (e.g., use Mn 2+ instead of Mg 2+ ) or adding chemical additives into reaction systems represent another directions to increase detection capability 25, 26 . Chemicals such as polyols, polymers, amino acid and their derivatives have been used as solvent additives to stabilize proteins and/or affect solvent properties, thereby influencing the kinetics of reaction chemistry 27 .…”