Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

Qiu, Miao; Zhou, Xiaoming; Liu, Lei

doi:10.1007/s41664-022-00212-4

Cited by 47 publications

(23 citation statements)

References 57 publications

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“…The kinetic rates quantified here are for buffer conditions and protocols typical of published CRISPR assays (see the SI). Despite the current work and significant work around optimization of conditions, ,,, the maximum possible kinetic rates for CRISPR systems are unknown. For example, trans -cleavage kinetic rates vary significantly among CRISPR orthologues and subtypes.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…Multiplexed approaches also reduce the need for template amplification, with larger arrays of crRNA targets diminishing the need for template amplification entirely [ 30 ]. After determining the most efficient path for crRNA design, we elected to not automate any modifications to the 3’ or 5’ ends of the identified crRNA targets, given the current debate on the effectiveness of such modifications [ 31 , 32 ].…”

Section: Discussionmentioning

confidence: 99%

PrimedSherlock: a tool for rapid design of highly specific CRISPR-Cas12 crRNAs

Mann

Pitts

2022

BMC Bioinformatics

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Background CRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest. Results Utilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise. Conclusions PrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.

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“…Moreover, Ma et al [ 58 ] discovered that the reaction buffer containing Mn 2+ was more suitable for the LbCas12a system, whereas the fluorescence signal generated by the reaction buffer containing Mg 2+ was 3.4–13.6 times greater. In addition to altering the divalent cations in the reaction system, additives like polyethylene glycol (PEG), glycerin, Triton X-100, bovine serum albumin (BSA), i-proline, etc., can increase the sensitivity of Cas12a in nucleic acid detection systems [ 59 , 60 ]. Cas12a nuclease generated sticky ends at the site of cleavage, whereas Cas12b nuclease cleavage resulted in a staggered break of 7 nucleotides at the cleavage site.…”

Section: Mechanism Of Molecular Detection Technology Based On Crispr/...mentioning

confidence: 99%

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection

Yan

Long

et al. 2023

Genes

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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

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Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

Cited by 47 publications

References 57 publications

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

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

PrimedSherlock: a tool for rapid design of highly specific CRISPR-Cas12 crRNAs

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection

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