COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Abstract

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene.

Evaluation of an automated CRISPR-based diagnostic tool for rapid detection of COVID-19

“…For example, the FnCas9 Editor Linked Uniform Detection Assay (FELUDA) was reported by Azhar et al, with a sensitivity of 100% and specificity of 97% [ 20 ]. Similar CRISPR methods has been developed and evaluated by Fasching et al [ 21 ], Mahas et al [ 22 ], and Liu et al [ 23 ]. CRISPR-based assays can also detect single-copy targets [ 24 ] and distinguish targets with single base differences [ 25 ] to permit sensitive and accurate mapping of SARS-CoV-2 RNA changes in respiratory and non-respiratory samples.…”

Uncertainty Quantification of Michaelis–Menten Kinetic Rates and Its Application to the Analysis of CRISPR‐Based Diagnostics

“…A recent and important example of Michaelis-Menten rate parameter evaluation is the quantification of CRISPR-associated (Cas) enzyme kinetics. CRISPR-Cas systems are used to detect nucleic acid sequences with high specificity, [13][14][15][16][17] and the limit of detection of such assays is directly governed by the kinetic rates of the enzyme. [18][19][20] The accurate quantification of the kinetic parameters is therefore paramount to evaluate the reliability and regime of applicability of CRISPR-based diagnostics assays.…”

Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a

“…Cas12a is sensitive to mismatches, but achieving single-nucleotide polymorphism (SNP) genotyping is still arduous, as mismatch(es) can decrease the signal but cannot abolish it ( 27 , 28 ). Current studies using CRISPR-Cas12a technology for detection of these variants include the following: (i) detection of Δ144, E484K, and N501Y with only Alpha variant differentiation ( 19 ); (ii) a high-fidelity Cas12 enzyme named CasDx1 that was tested on L452R, E484K, and N501Y but cannot be used for variant differentiation due to emergence of the Omicron variant ( 29 ); and (iii) two platforms based on reverse transcription PCR (RT-PCR) and CRISPR-Cas12a, which rely on a fluorescence reader to differentiate variants because the wild type and other mutations at the same sites showed false-positive signals, and thus, tube visualization or lateral flow strip are not applicable ( 18 , 30 ).…”

“…Although Cas12a and crRNAs can be engineered to achieve higher specificity, crRNA modification is relatively easier and safer ( 27 – 29 , 31 ). Four crRNA designing strategies were employed to improve the specificity of crRNAs in this study, which include (i) chimeric crRNA (replacing the last 8 nucleotide [nt] RNA of the 24-nt spacer with DNA) ( 28 , 32 ), (ii) short crRNA (15-nt to18-nt spacer) ( 33 ), (iii) 3'DNA7 (7-nt DNA 3′ end extension to crRNA, spacer + 3′ end 7 universal DNA extension [ TATTATT ]) ( 27 ), and (iv) introduction of one or two mismatches in the spacer of crRNA.…”