SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

Steens, Jurre A.; Zhu, Yifan; Taylor, David W.; Bravo, Jack P.K.; Prinsen, Stijn H.P.; Schoen, C.D.; Keijser, Bart J. F.; Ossendrijver, Michel; Hofstra, L. Marije; Brouns, Stan J. J.; Shinkai, Akeo; Oost, John van der; Staals, Raymond H.J.

doi:10.1038/s41467-021-25337-5

Cited by 67 publications

(98 citation statements)

References 73 publications

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“…It was observed that 40% of the studies were conducted in the USA only [ 14 , 15 , 25–28 ], 13% in Canada [ 29 , 17 ] and China [ 2 , 30 ] each. Apart from this, some studies have been conducted in Germany [ 31 ], Singapore (Ooi et al, 2021[ 32 ]), Netherlands [ 18 ], Japan [ 33 ], and Saudi Arabia [ 34 ], representing 7% each from included studies ( Figure 2 ). Patient’s details have not been specified in any of the articles, although the type of specimen taken for most of the studies was nasopharyngeal swab (n = 10) [ 14 , 25 , 26 , 2 , 13 , 17 , 18 , 27 , 28 , 31 ].…”

Section: Resultsmentioning

confidence: 99%

Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis

Bhatt

Bumbrah

Ruwali

et al. 2022

Pathogens and Global Health

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To address the challenges associated with COVID-19 diagnosis, we need a faster, direct, and more versatile detection method for efficient epidemiological management of the COVID-19 pandemic. RT-qPCR (reverse transcription quantitative real-time Polymerase Chain Reaction) although the most popular diagnostic method suffers from a major drawback of equipment dependency and trained molecular biologists that limits rapid and large-scale screening, particularly in low resource regions. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a feasible alternative for RT-qPCR; however, it also suffers from the drawback of false-positive issues. Recently, RT-LAMP has been integrated with the CRISPR-Cas technique to take care of the problems associated with RT-LAMP for COVID-19 diagnosis. In this study, a meta-analysis was conducted using three scientific databases considering the PRISMA guidelines to assess the diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technology. Out of a total of 1286 studies on COVID-19, we identified 15 articles that met our eligibility criteria of using simultaneous RT-LAMP and CRISPR-Cas technique. Our meta-analysis of the included studies revealed that most of the studies were conducted in the USA with the N gene as the most common target and fluorescence-based detection method. The meta-analysis results of all included studies have further revealed a pooled sensitivity value of higher than 85% and a pooled specificity value of 80% with the confidence interval of 95%, respectively, as revealed from the forest plot and SROC curve. The accuracy rate of included studies was also calculated which varied from 77.4% to 100%. Furthermore, the precision of included studies varied from 75% to 100%. Lastly, a quality assessment of bias and applicability was performed based on QUADAS-2. Taken together, combined RT-LAMP and CRISPR-Cas technique could be a potential alternative to RT-qPCR particularly in low resource regions having a high demand for rapid testing.

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Section: Resultsmentioning

confidence: 99%

Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis

Bhatt

Bumbrah

Ruwali

et al. 2022

Pathogens and Global Health

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“…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%

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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“…An example of such apparently incomplete system is the Type III-B CRISPR-Cas system of Thermus thermophilus , whose Cas10 protein lacks the HD domain [ 43 ]. However, since Cas10 possesses an intact Palm domain, it is still capable of activating auxiliary effectors via the cOAs pathway [ 44 ]. The Type III-B locus of T. thermophilus encodes a cOA-activated DNA nickase, which might be responsible for the plasmid degradation in the absence of the Cas10 HD DNase activity [ 45 ].…”

Section: History Of Type III Crispr-cas Researchmentioning

confidence: 99%

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

Kolesnik

Fedorova

Karneyeva

et al. 2021

Biochemistry Moscow

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The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit – the so-called CRISPR polymerase – which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

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SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

Cited by 67 publications

References 73 publications

Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis

Diagnostic efficiency of RT-LAMP integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis

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

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

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