A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120–145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

[1]  A. Zherdev,et al.  Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors , 2023, Biosensors.

[2]  A. Zherdev,et al.  Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors , 2023, International journal of molecular sciences.

[3]  X. Le,et al.  Signal Amplification by the trans-Cleavage Activity of CRISPR-Cas Systems: Kinetics and Performance , 2023, Analytical chemistry.

[4]  Xiaoping Zhou,et al.  Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives. , 2023, Chemical Society reviews.

[5]  R. Aman,et al.  CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications , 2022, ACS synthetic biology.

[6]  Xianlong Zhang,et al.  CRISPR/Cas Systems‐Inspired Nano/Biosensors for Detecting Infectious Viruses and Pathogenic Bacteria , 2022, Small methods.

[7]  Jingyun Li,et al.  Amplification-Free, Single-Microbead-Based Cas12a Assay for One-Step DNA Detection at the Single-Molecule Level. , 2022, Analytical chemistry.

[8]  C. Mirkin,et al.  Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters. , 2022, Journal of the American Chemical Society.

[9]  H. Nishimasu,et al.  Automated amplification-free digital RNA detection platform for rapid and sensitive SARS-CoV-2 diagnosis , 2022, Communications Biology.

[10]  A. Zherdev,et al.  Comparative study of magnetic beads and microplates as supports in heterogeneous amplified assay of miRNA-141 by using mismatched catalytic hairpin assembly reaction. , 2022, Talanta.

[11]  An-Na Tang,et al.  MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a , 2022, Chemical science.

[12]  A. Zherdev,et al.  DIRECT2: A novel platform for a CRISPR-Cas12-based assay comprising universal DNA-IgG probe and a direct lateral flow test. , 2022, Biosensors & bioelectronics.

[13]  Lei Liu,et al.  Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection , 2022, Journal of Analysis and Testing.

[14]  Minyi Liu,et al.  Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system. , 2022, The Analyst.

[15]  Amina Antonacci,et al.  Isothermal amplification-assisted diagnostics for COVID-19 , 2022, Biosensors and Bioelectronics.

[16]  E. Osdaghi.  Dickeya solani (black leg disease of potato) , 2022, CABI Compendium.

[17]  G. Saddler.  Dickeya solani (black leg disease of potato). , 2022, PlantwisePlus Knowledge Bank.

[18]  J. Wang,et al.  Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics , 2021, Frontiers in Microbiology.

[19]  Zifan Tang,et al.  Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores , 2021, Nano letters.

[20]  P. Sax,et al.  Clinical and Economic Effects of Widespread Rapid Testing to Decrease SARS-CoV-2 Transmission , 2021, Annals of Internal Medicine.

[21]  G. Khan,et al.  CRISPR-Cas12-based nucleic acids detection systems. , 2021, Methods.

[22]  D. Xing,et al.  Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity. , 2020, Chemical communications.

[23]  Nicolaas M. Angenent-Mari,et al.  Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release , 2020, Nature Protocols.

[24]  Lingwen Zeng,et al.  An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids. , 2020, Biosensors & bioelectronics.

[25]  A. Zherdev,et al.  Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani. , 2020, Molecular and cellular probes.

[26]  Juan G. Santiago,et al.  Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2 , 2020, Proceedings of the National Academy of Sciences.

[27]  Guixia Yu,et al.  CRISPR-Cas12–based detection of SARS-CoV-2 , 2020, Nature Biotechnology.

[28]  G. Montoya,et al.  CRISPR-Cas12a: Functional overview and applications , 2020, Biomedical journal.

[29]  Shiyuan Li,et al.  HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. , 2019, ACS synthetic biology.

[30]  James J. Collins,et al.  Programmable CRISPR-responsive smart materials , 2019, Science.

[31]  Qi Zhou,et al.  CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity , 2019, Genome Biology.

[32]  Kornel Labun,et al.  CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing , 2019, Nucleic Acids Res..

[33]  D. C. Swarts,et al.  Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a. , 2019, Molecular cell.

[34]  Johannes Thomsen,et al.  Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity , 2018, Cell.

[35]  In‐San Kim,et al.  Direct observation of DNA target searching and cleavage by CRISPR-Cas12a , 2018, Nature Communications.

[36]  James R. Rybarski,et al.  Kinetic basis for DNA target specificity of CRISPR-Cas12a , 2018, bioRxiv.

[37]  Jennifer A. Doudna,et al.  CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity , 2018, Science.

[38]  Shiyuan Li,et al.  CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA , 2018, Cell Research.

[39]  S. Keten,et al.  Persistence length and stochastic fragmentation of supramolecular nanotubes under mechanical force , 2013, Nanotechnology.

[40]  J. Remacle,et al.  Comparison between microwell and bead supports for the detection of human cytomegalovirus amplicons by sandwich hybridization. , 1997, Analytical biochemistry.

[41]  P. Hagerman,et al.  Investigation of the flexibility of DNA using transient electric birefringence , 1981, Biopolymers.

[42]  Guo-Ping Zhao,et al.  CRISPR-Cas12a-assisted nucleic acid detection , 2018, Cell Discovery.

[43]  G. Frens.  Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions , 1973 .