A chemical-enhanced system for CRISPR-Based nucleic acid detection

Abstract

The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

“…Multiple studies have shown that when combining RT-LAMP/RT-RPA with the CRISPR reaction, the sensitivity of detection is reduced significantly, possibly due to operating temperature differences, an inhibitory effect of excessive amount of amplified product, differences in optimal buffer and salt conditions, and unwanted nonspecific trans-cleavage of Cas effector on primers. 1 , 17 , 18 , 19 , 20 However, having observed such a robust trans-cleavage with a small delay after amplification, we consider that BrCas12b has circumvented many of these issues.…”

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

“…Polyethylene glycol (PEG), glycerin and Triton X-100 can improve the sensitivity of Cas12a system, but their mechanism of promoting trans-cleavage activity is unknown [ 16 ]. Li et al [ 17 ] found that bovine serum albumin (BSA) could improve sensitivity of Cas12a system, while L-proline could protect Cas12a from an adverse environment. When BSA was added together with L-proline, L-proline improved sensitivity in the way of protecting BSA and Cas12a.…”

RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

“…The clinical sensitivity and specificity of the RT-LAMP CRISPR-based SARS-CoV-2 detection methods were in the ranges of 75–100% and 95.5–100%, respectively ( Table 1 ). Several strategies for improving the sensitivity and specificity of RT-LAMP CRISPR-based SARS-CoV-2 detection have been explored, such as the utilization of multiple crRNAs, optimization of the RT-LAMP temperature and reaction time [ 28 , 43 ], modification of the crRNA [ 25 ], optimization of the length of the reporter probe [ 6 ], and addition of bovine serum albumin and L-proline to Cas12/Cas13-based detection reactions [ 44 ].…”