Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

Santiago-Frangos, Andrew; Hall, Laina N.; Nemudraia, Anna; Nemudryi, Artem; Krishna, Pushya; Wiegand, Tanner; Wilkinson, Royce A.; Snyder, Deann T.; Hedges, Jodi F.; Cicha, Calvin; Lee, Helen H.; Graham, Ava; Jutila, Mark A.; Taylor, Matthew P.; Wiedenheft, Blake

doi:10.1016/j.xcrm.2021.100319

Cited by 60 publications

(76 citation statements)

References 50 publications

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“…Indeed, wide variations in the sensitivity of the Cas13 effector have been observed with different guide RNA sequences during SARS-CoV-2 assay development ( 38 ). In contrast, a recent study from the Wiedenheft lab ( 40 ) detected only minor differences in the sensitivity of 10 guides designed to detect different regions of the N gene using a type III effector. A notable difference is that our assays were carried out at 37°C whereas the Wiedenheft group used a thermostable TtCsm complex active at 60°C.…”

Section: Discussionmentioning

confidence: 87%

“…The combination of VmeCmr and NucC with an optimal guide RNA and fluorescence readout provided a LoD of 2 fM for detection of the SARS-CoV-2 N gene transcript, consistent with the observation that a reaction with fewer than 1 × 10 5 copies of the viral RNA (8 fM) can be readily detected (Figure 8 ). This compares with a LoD using the T. thermophilus Csm (TtCsm) system of 1 × 10 7 copies (100 pM) ( 40 ) and 1.2 × 10 8 copies (1 nM) detected by the TtCmr system ( 41 ). The >100-fold higher sensitivity of the assay we describe here is likely due to a combination of factors.…”

Section: Discussionmentioning

confidence: 99%

“…In the past few years, the collateral cleavage activities of Cas12 and Cas13 have been repurposed for the development of rapid and specific assays capable of detecting specific RNA or DNA sequences ( 36–39 ). Two recent studies have demonstrated that the ability of type III (Cas10) CRISPR systems to detect RNA specifically and synthesise cOA can be harnessed in combination with CARF family effector nuclease Csm6 to provide an alternative assay system ( 40 , 41 ). Here, we demonstrate that a prophage-encoded Vibrio type III-B system generates cA 3 on specific target RNA binding, resulting in the activation of the NucC effector nuclease for non-specific dsDNA degradation.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Grüschow

Adamson

White

2021

Nucleic Acids Research

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Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Grüschow

Adamson

White

2021

Nucleic Acids Research

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“…By combining isothermal amplification methods (e.g., Recombinase Polymerase Amplification, RPA (Piepenburg et al, 2006), or Loop-mediated isothermal AMPlification (LAMP) (Notomi et al, 2000) with reporting formats such as lateral flow detection with antigen-labeled reporters (Kellner et al, 2019), DETECTR (Broughton et al, 2020;Ding et al, 2020;Lucia et al, 2020) and SHERLOCK (Ackerman et al, 2020;Metsky et al, 2020) have recently been used for rapid and highly sensitive SARS-CoV-2 detection. Furthermore, during the peer reviewing period of this manuscript, type III CRISPR-Cas systems have also been reported for SARS-CoV-2 diagnosis (Santiago-Frangos et al, 2021). The Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), has also been shown to inhibit and degrade SARS-CoV-2 viral RNA in respiratory epithelial cells (Abbott et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

et al. 2022

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CRISPR-based diagnostics (CRISPR-dx), including the Cas12-based DETECTR and Cas13-based SHERLOCK Class 2 CRISPRs, have been used to detect the presence of DNA or RNA from pathogens, such as the 2009 pandemic influenza virus A (IAV) and the 2019 novel coronavirus SARS-CoV-2. Here, we describe the collateral single-stranded DNA cleavage with Class 1 type I CRISPR-Cas3 and highlight its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This assay, which we call Cas3-Operated Nucleic Acid detectioN (CONAN), not only detects SARS-CoV-2 in clinical samples, but also offers specific detection of single-base-pair mutations in IAV variants. This tool allows rapid and accurate point-of-care testing for patients with suspected SARS-CoV-2 or drug-resistant IAV infections in hospitals.

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“…Systems that do not rely on nucleic acid pre-amplification have also been reported (Fozouni et al, 2021). Very recently, CRISPR/Cas type III systems have been harnessed for the development of pathogen detection platforms, thereby expanding the CRISPR/Cas diagnostic toolbox (Santiago-Frangos et al, 2021;Steens et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

iSCAN-V2: A One-Pot RT-RPA–CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection

Aman

Maršić

Rao

et al. 2022

Front. Bioeng. Biotechnol.

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Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

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Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

Cited by 60 publications

References 50 publications

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus

iSCAN-V2: A One-Pot RT-RPA–CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection

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