CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation

Abstract

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

“…The trans -cleavage activity of Cas12a facilitates the cleavage of reporter probes and generates amplified signals. The sensitivity of these assays is further improved by integrating CRISPR-Cas12a with nucleic acid amplification techniques, including PCR [ [134] , [135] , [136] , [137] ], recombinant polymerase amplification (RPA) [ 27 , 107 , 138 ], and loop-mediated isothermal amplification (LAMP) [ 34 , 139 ]. The viral RNA is reverse transcribed, the complementary DNA is amplified, and the amplicons are subsequently detected ( Fig.…”

“…This strategy has been used to develop CRISPR-Cas12a-based methods for the detection of SARS-CoV-2 variants. For example, Marqués et al [ 135 ] developed a CRISPR-Cas12a-based assay for the detection of the E484K mutation. E484K mutation, first identified in the Beta and Gamma variants, is a substitution of guanine by adenine in the S gene, leading to three consecutive adenines (WT 5′-TGAA-3′ and mutant 5′-TAAA-3′), which forms a canonical PAM sequence on the antisense strand (WT 5′-TTCA-3′ and mutant 5′-TTTA-3′).…”

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…497 Currently the available protocols cannot discriminate between specic VOCs/VOIs; however, RT-LAMP technology offers certain advantages such as faster test results and requirement of less resources, while maintaining high sensitivity and specicity. CRISPR-based variant detection techniques focus on designing crRNAs with mismatches for mutation positions in known sequences 498,499 or developing chimeric crRNAs to distinguish mutations in different positions specically. 500 Most of these technologies use uorescence 501 or LFAs 502 for the readout of the detection results, and some have integrated technology into micro devices to develop POCT devices that integrate extraction, purication, and concentration of viral RNA, amplication and detection.…”

CRISPR-associated type V proteins as a tool for controlling mRNA stability inS. cerevisiaesynthetic gene circuits

“…The CRISPR-Cas system, an RNA-based immune system in bacteria and archaea, has become a popular instrument for genome editing ( 8–10 ), disease diagnosis ( 11 , 12 ), and gene therapy ( 13 ). To date, two classes and six types of CRISPR-Cas systems are known.…”