A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance

Abstract

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection—such as multiplexed detection for viral variant surveillance—may limit their widespread adoption. Herein, we developed a robust multiplexed CRISPR-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose SARS-CoV-2 infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)—including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)—all while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool—CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance which can be locally manufactured—may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

“…Several studies have developed methods to distinguish among different strains of SARS-CoV-2. Isothermal amplification techniques such as Recombinase Polymerase Amplification (RPA), 15,16 Recombinase-Aided Amplification (RAA), 17 and Loop-mediated Isothermal Amplification (LAMP) 18 are rapid and simple methods that can be used for POC detection. On the contrary, there are some limitations of isothermal amplification methods, such as the secondary structure of primers (longer than 30 bp for RPA and RAA), complicated primer design (at least three primer pairs for LAMP), optimized primer concentration, suitable incubation temperature, and downstream detection method.…”

CRISPR‐Cas system: A promising tool for rapid detection of SARS‐CoV‐2 variants

“…Speed: The current CRISPR technology platform can complete the detection process in approximately 1 h. Simplicity: The method, when combined with isothermal amplification, eliminates the need for thermal cyclers. Moreover, the development of freeze‐dried reagents enables easy access in economically disadvantaged areas 80 . Researchers have also developed a one‐pot reaction, consolidating multiple steps into a single process, thereby increasing the user‐friendliness of CRISPR‐based detection. Affordability: The detection cost of the Specific High‐sensitivity Enzymatic Reporter unLOCKing version 2 (SHERLOCKv2) test strip is only a few dollars, 43 while the DETECTR test costs less than one dollar per test 45 …”

“…Simplicity: The method, when combined with isothermal amplification, eliminates the need for thermal cyclers. Moreover, the development of freeze‐dried reagents enables easy access in economically disadvantaged areas 80 . Researchers have also developed a one‐pot reaction, consolidating multiple steps into a single process, thereby increasing the user‐friendliness of CRISPR‐based detection.…”

Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis

“…13 On the other hand, the CRISPR/Cas12a system could boost the specificity of RPA, and the sequence-specific recognition of the CRISPR system could reduce false-positive results caused by the RPA products. 14 amplify the target DNA at a constant temperature, and the amplified DNA is transferred to the CRISPR/Cas12a system. The Cas12a protein is activated by the target sequence, which cleaves the amplified product subsequently and the fluorescent reporter, generating a fluorescence signal.…”

“…The RPA assay could improve the sensitivity of the CRISPR/Cas12a assay . On the other hand, the CRISPR/Cas12a system could boost the specificity of RPA, and the sequence-specific recognition of the CRISPR system could reduce false-positive results caused by the RPA products . DETECTR uses the RPA system to amplify the target DNA at a constant temperature, and the amplified DNA is transferred to the CRISPR/Cas12a system.…”