Sensitive and multiplexed RNA detection with Cas13 droplets and kinetic barcoding

Son, Sungmin; Lyden, Amy; Shu, Jeffrey; Stephens, Stephanie I.; Fozouni, Parinaz; Knott, Gavin J.; Smock, Dylan C. J.; Liu, Tina Y.; Boehm, Daniela; Simoneau, Camille R.; Kumar, Renuka; Doudna, Jennifer A.; Ott, Mélanie; Fletcher, Daniel A.

doi:10.1101/2021.08.02.21261509

Cited by 12 publications

(20 citation statements)

References 29 publications

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“…Our engineered CRISPR-Cas system is among one of the most sensitive CRISPR-based amplification-free technologies for the detection of nucleic acid developed to date in detecting the SARS-CoV-2 genome (LoD 0.6 copies per microliter in the reaction, within 30 min). It shows comparable sensitivity to the droplet-based Cas13a kinetic barcoding (below 1 copy per microliter), which contained a total of 26 crRNAs in one single assay 49 ; it is one order of magnitude more sensitive than that of the Cas13a-Csm6 tandem assay (31 copies per microliter), which required eight different crRNAs and a Csm6 protein with its chemically modified activator RNA 17 ; two orders of magnitude more sensitive than the Cas13a-mobile phone microscopy (200 copies per microliter), which utilized three different crRNAs 16 ; three orders of magnitude more sensitive than the Cas13-microchamber array 14 , and six orders of magnitude more sensitive than the CRISPR-electrochemical biosensor that depends on the WT Cas13a protein 18 . As opposed to the aforementioned amplification-free detection technologies that are exclusively based on extracted RNA materials, our RBD-LwaCas13a-electrochemistry platform showed great detection sensitivity and accuracy and does not need the additional RNA extraction steps, which holds great potential for ultrasensitive detection of various RNAs of clinical and environmental importance.…”

Section: Discussionmentioning

confidence: 97%

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

et al. 2022

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Cas13 orthologs, the single effectors of the Class 2 type VI clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems, are RNA-guided ribonucleases 1 . The CRISPR RNAs (crRNAs) of this family contain a direct repeat stem loop that interacts with the Cas13 protein to form an RNase-inactive binary complex and a spacer sequence that base pairs with the target RNA 2 . The resulting Cas13-crRNA-target ternary complex undergoes a large-scale conformational change in which two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains move toward each other to form a single catalytic pocket to cleave the target RNA. Intriguingly, this catalytic pocket localized on the outer surface of the target-activated Cas13 complex can non-specifically cleave any surrounding RNA molecules in a characteristic 'collateral effect' 2 . This target-triggered collateral activity, originally an immune defense mechanism intended to induce host dormancy and prevent the propagation of invading phages, has been rapidly developed for in vitro detection of nucleic acids 3 . For example, the Cas13-crRNA surveillance complex is activated upon target recognition and performs collateral cleavage of nearby dye-quencher pairs linked by single-stranded RNA (ssRNA) 3,4 to rapidly generate measurable fluorescent or colorimetric signals [3][4][5] .The Leptotrichia wadei (Lwa)Cas13a has been widely used in nucleic-acid-based diagnostics. Coupling LwaCas13a collateral activity to target preamplification, the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform has detected attomolar levels of viral RNA and other endogenous RNA targets 3 . Combining LwaCas13a with auxiliary Csm6 endoribonuclease, SHERLOCKv2 shortened the detection time for a Dengue

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Section: Discussionmentioning

confidence: 97%

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

et al. 2022

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“…69 They showed that the LOD was improved 100-fold with the same CRISPR reaction time. In another study, Son et al 72 utilized 26 different crRNAs in a Cas13a assay and improved the LOD 5 times. It is clear that utilizing multiple crRNAs could decrease the FOM value and improve the system performance.…”

Section: ■ Fom Improvement Strategiesmentioning

confidence: 99%

“…It is noteworthy that multiple strategies could be implemented in one system to achieve lower FOM compared to individual strategies. For instance, Son et al 72 combined digitalization and multiple crRNA in a single system and reduced the FOM by more than 6 orders of magnitude compared to the nonamplified Sherlock system. 9 To guide the implementation of improvement strategies for different applications, we summarized the advantages and disadvantages of each strategy in Table 1.…”

Section: ■ Fom Improvement Strategiesmentioning

confidence: 99%

“…39,70,85 The best FOM performance was observed by combining digital assay and multiple crRNA cases, where FOM deceased to 24 aM•min. 72 As a result, digital CRISPR assay provides the most appealing method for amplification-free CRISPR-based nucleic acid sensing. Since digital CRISPR-based sensing is a new trend, limited data is available and more studies in the future would improve this evaluation.…”

Section: ■ Performance Benchmarkingmentioning

confidence: 99%

“…It is noteworthy that multiple strategies could be implemented in one system to achieve lower FOM compared to individual strategies. For instance, Son et al . combined digitalization and multiple crRNA in a single system and reduced the FOM by more than 6 orders of magnitude compared to the nonamplified Sherlock system …”

Section: Fom Improvement Strategiesmentioning

confidence: 99%

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Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

et al. 2022

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Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs, digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.

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Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics

Huyke

Ramachandran

Bashkirov³

et al. 2022

Anal. Chem.

110

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Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis−Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 10 5 and 10 6 M −1 s −1 . For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.

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Sensitive and multiplexed RNA detection with Cas13 droplets and kinetic barcoding

Cited by 12 publications

References 29 publications

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics

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