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

Fasching, Clare L.; Servellita, Venice; McKay, Bridget; Nagesh, Vaishnavi; Broughton, James P.; Brazer, Noah; Wang, Baolin; Sotomayor-González, Alicia; Reyes, Kevin; Streithorst, Jessica; Deraney, Rachel N.; Stanfield, Emma; Hendriks, Carley G; Miller, Steve; Ching, Jesus; Chen, Janice S.; Chiu, Charles Y.

doi:10.1101/2021.11.29.21267041

Cited by 7 publications

(9 citation statements)

References 53 publications

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“…In the current version of CriSNPr, six of the widely used CRISPR diagnostic platforms have been included due to enough literature support for their design guidelines. The field of CRISPRDx being rapidly evolving, several other Cas proteins have been recently reported for detecting pathogenic DNA or RNA and their associated variants (66,67). As more and more literature supporting these diagnostic pipelines become available, we would include them in the server.…”

Section: Discussionmentioning

confidence: 99%

CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems

Chakraborty

Ansari

Kumar

et al. 2022

Preprint

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Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facilitated clinical decision-making, particularly during the COVID-19 pandemic. This has been further accelerated through the discovery of newer and engineered CRISPR effectors, expanding the portfolio of such diagnostic applications to a wide variety of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline requires customized detection schemes originating from fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is particularly relevant for variant detection, an attractive low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. In addition, we provide a database of curated pre-designed gRNAs and target/off-target for all human and SARS-CoV-2 variants reported so far. CriSNPr has been validated on multiple Cas proteins and highlights its broad and immediate scope of utilization across multiple detection platforms. CriSNPr is available at URL http://crisnpr.igib.res.in/.

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Section: Discussionmentioning

confidence: 99%

CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems

Chakraborty

Ansari

Kumar

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…There have been several reports of CRISPR detection of SNPs in the literature. In particular, Cas12a, 10,13 Cas13a, 7,9,11,12,14 and Cas14a 15 have been used to confirm that CRISPR-based assays are sufficiently specific for SNP detection. For example, Shan et al 14 showed differences in initial reaction velocities for Cas13 when activated by WT versus SNPs within six bases of the target 5′ end but did not consider the effects of SNPs on Michaelis−Menten kinetics.…”

Section: ■ Introductionmentioning

confidence: 99%

Detection and Discrimination of Single Nucleotide Polymorphisms by Quantification of CRISPR-Cas Catalytic Efficiency

et al. 2022

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The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., cancer and SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable kinetic shifts in the Cas12 trans-cleavage substrate affinity (K M ) and apparent catalytic efficiency (k cat * /K M ). To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michaelis−Menten parameters and apply this assay to a 20 base pair WT target of the SARS-CoV-2 E gene. We find that the k cat * /K M measured for WT is 130-fold greater than the lowest k cat * /K M among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). K M also offers a strong ability to distinguish SNPs, varies 27-fold over all the cases, and, importantly, is insensitive to the target concentration. Last, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.

show abstract

Section: Introductionmentioning

confidence: 99%

“…There have been several reports of CRISPR detection of SNPs in the literature. In particular, Cas12a, 10,13 Cas13a, 7,9,11,12,14 and Cas14a 15 have been used to confirm that CRISPRbased assays are sufficiently specific for SNP detection. For example, Shan et al 14 showed differences in initial reaction velocities for Cas13 when activated by WT versus SNPs within 6 bases of the target 5' end but did not consider effects of SNPs on Michaelis-Menten kinetics.…”

Section: Introductionmentioning

confidence: 99%

Detection and discrimination of single nucleotide polymorphisms by quantification of CRISPR-Cas catalytic efficiency

Blanluet

Huyke

Ramachandran

et al. 2022

Preprint

View full text Add to dashboard Cite

The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency . To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michalis-Menten parameters and apply this assay to a 20-base pair WT target of the SARS-CoV-2 E gene. We find that the measured for WT is 130-fold greater than the lowest among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers strong ability to distinguish SNPs, varies 27-fold over all the cases, and is insensitive to target concentration. Lastly, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.

show abstract

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

Cited by 7 publications

References 53 publications

CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems

CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems

Detection and Discrimination of Single Nucleotide Polymorphisms by Quantification of CRISPR-Cas Catalytic Efficiency

Detection and discrimination of single nucleotide polymorphisms by quantification of CRISPR-Cas catalytic efficiency

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