key: cord-0332000-liftb1iu authors: Liu, F. X.; Cui, J. Q.; Park, H.; Chan, K. W.; Leung, T.; Tang, B. Z.; Yao, S. title: Microfluidics-Enabled Digital Isothermal Cas13a Assay date: 2021-08-24 journal: nan DOI: 10.1101/2021.08.18.21262201 sha: 16f38fad0aca19c5ae8b957119c454a2d8166c5e doc_id: 332000 cord_uid: liftb1iu The isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint (qPCR) has long been regarded as a gold standard for viral infection diagnosis 2 . However, as a 44 centralized laboratory-based testing paradigm for viral diagnostics, PCR assays depend on 45 specifically designed TaqMan probes as well as precise thermal cycling, limiting their 46 deployability for community tests. 47 Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR- SHERLOCK can perform at a one-pot scheme, the macromolecular crowding agent issue and 60 enzymatic incompatibility between RPA and CRISPR reactions result in less robust and sensitive 61 results compared to two-step reactions 17 . In addition, the one-pot reaction relies on the use of 62 standard curves and endogenous controls to obtain relative quantification, which has been shown 63 to have significant variations 18, 19 . In contrast to the standard curve calibration approach, absolute 64 quantification with improved precision and accuracy has been achieved by digital PCR 20 and 65 digital loop-mediated isothermal amplification (LAMP) 21 . In digital quantification, the assay 66 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint mixture is partitioned into a sufficient number of individual reactions so that either zero or one of 67 the target nucleic acid molecules is being amplified in each partition, and the assay result is 68 evaluated based on a simple positive-negative counting of the individual partitions based on the 69 end-point fluorescence measurement 19 . Yet, as far as we know, there is no digital SHERLOCK 70 assay available. 71 Different from PCR or LAMP that needs to be triggered by elevating the temperature, the 72 isothermal RPA and some of the CRISPR type V and VI endonucleases can react at room 73 temperature 22-24 so that the reaction would start to amplify the targets before the sample 74 partitioning into the nano-wells (e.g., QuantStudio 3D digital chips) or droplets using a simple 75 flow-focusing system, which inevitably causes undesired overestimation due to pre-amplification 76 24-26 . To minimize the premature target amplification, one remedy is to prepare the reaction 77 mixture on ice and quickly load it for partition 27 . Hence, unlike hot-start digital PCR and LAMP, 78 the implementation of digital quantification of the near ambient temperature SHERLOCK needs a 79 novel system for sample partitioning. Droplet microfluidics offers versatile yet controllable 80 handling of samples and reagents in forming droplets, and thus can effectively avoid premature 81 amplification before partitioning into droplets as well as facilitate combined amplification and 82 trans-cleavage detection reactions in individual droplets. In this work, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), the 84 digital format of enhanced one-pot SHERLOCK (E-SHERLOCK). Digital quantification relies on 85 a high signal-to-background ratio to convert the fluorescent signals of individual droplets to one 86 or zero readouts, thus signal robustness and reproducibility are of great importance for digital 87 quantification. However, when we conducted the one-pot SHERLOCK assay, the poor sensitivity 88 and robustness of the reaction raised great concern. We identified the optimal reaction condition 89 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Overview of MEDICA for viral HPV Detection. 105 We established the workflow of MEDICA and demonstrated its application for the detection of for the most precancerous lesions 28 . We designed two MEDICA sequences that include two sets 109 of forward primers with overhanging T7 promoter, Cas13a crRNAs to recognize both HPV 16 and 110 HPV 18 for specific and sensitive tests (Figure 1a ). RPA is a magnesium acetate (MgOAc) 111 initiation nucleic acid amplification near ambient temperature compared to hot start LAMP and 112 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint Optimization of the one-pot SHERLOCK assay 146 Combining RPA with Cas13a in a one-pot reaction with high sensitivity and reproducibility is 147 critical to ensure the accuracy and robustness of the quantification results. Therefore, we started 148 with assay development which is a prerequisite to digital quantification. However, when we 149 conducted the one-pot assay directly combining the reagents used in the two-step SHERLOCK 35 , Subsequently, we performed multiple rounds of assay optimization to identify the key factors of 160 this reaction. First, we investigated the components of the reaction buffer including pHs, salts, and 161 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint other additives. We found the 60 mM pH 7.5 Tris-HCl buffer with 80 mM KCl yielded the highest 162 signal, much better than the HEPES buffer used for SHERLOCK detection (Figure 2b and 163 supplementary S3a). The commonly used RPA mixture is highly viscous due to the presence of a 164 high molecular crowding agent to enhance the catalytic activity and increase specificity as well as (Table 1) . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint Additionally, the droplet digital technique offers more precise quantification results compared to 229 real-time quantitative detection. Therefore, we applied droplet microfluidics to covert our optimal 230 assay into digital format. The droplet digital assay contains three steps, droplet partition, droplet incubation, and digital Figure 3e shows the fluorescence intensity from the NTC group, which was 259 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint Detailed information about reagents, including the commercial vendors and stock concentrations, 360 all chemicals and sequences related to this work are listed in supplementary table 4 and table 5 . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint was dialyzed in 2 L of 1X PBS in a cellulose membrane dialysis tube for 18 h. The presence of 383 recombinant was identified using SDS-PAGE gel analysis (Supplementary S1). Amicon Ultra-0.5 384 mL centrifugal filter was used to concentrate protein and re-diluted in protein storage buffer, which 385 allows the protein for storage at -80 °C for up to 6 months. The two-step SHERLOCK is divided into two aspects: First, the RPA pre-amplification was 411 triggered by adding MgOAc from the kit instructed with TwistAmp® Basic at 37 °C for 20 min. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint sample or viral specimen was pipetted into this master mix with thorough mixing. Finally, 1μL of 427 MgOAc (280 mM) from the kit was added to trigger this reaction. Then optimization occurred iteratively with each reagent modification. We identified each 429 component to yield the best signal then kept the optimal reagent tested previously when conducted . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint We further evaluated the performance of the sensitivity, specificity, and comparison of this one-448 pot reaction. All the experiments were conducted in real-time with the Roch Lightcycler 480 II 449 with fluorescence measurement every minute at 37 °C for 1 h. For the specificity test, the spiked 450 genes were firstly prepared at 1 × 10 5 cp/µL and mixed mutually before experiments. is suitable for water-in-oil droplets formation. After binding, the microfluidic chip was firstly pre-470 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint treated with aquapel (PPG Industries) to yield hydrophobicity and followed by fluorinated Novec 471 7100 (3M) washing for 30 s. The microfluidic chip was incubated at a hotplate at 80 °C before use. The experiment was conducted with a pressure box (Everflow) to control the three phases 474 (magnesium phase, master mix with target and oil phase). The total reaction volume is 20 μL The droplet imaging system includes a LED light source with a 488 nm filter and a Nikon 491 microscope. All the information (number and fluorescence intensity) was calculated by ImageJ 492 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint A novel coronavirus outbreak of global 517 health concern Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-519 qPCR primer-probe sets Massively multiplexed nucleic acid detection with Cas13 Streamlined inactivation, amplification, and Cas13-based detection of 523 SARS-CoV-2 Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola 525 and Lassa virus cases in real-time CRISPR-Cas12-based detection of SARS-CoV-2 Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual 529 CRISPR-Cas12a assay International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted Detection of SARS-CoV-2 with SHERLOCK one-pot testing Clinical validation of a Cas13-based assay for the detection of SARS-535 CoV-2 RNA CRISPR-Cas guides the future of genetic engineering Next-generation diagnostics with CRISPR Nucleic acid detection with CRISPR-Cas13a/C2c2 CRISPR-Cas12a target binding unleashes indiscriminate single-stranded 542 RNA 544 targeting by functionally orthogonal type VI-A CRISPR-Cas enzymes CRISPR Enzyme Kinetics for Molecular Diagnostics International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted Viral diagnostics in the era of digital polymerase chain 551 reaction Absolute quantification by droplet digital PCR versus analog real-time Digital PCR-an emerging technology with broad 555 applications in microbiology Digital loop-mediated 557 isothermal amplification on a commercial membrane A portable reverse transcription recombinase polymerase 559 amplification assay for rapid detection of foot-and-mouth disease virus An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides 562 enable robust and rapid COVID-19 testing Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of 564 SARS-CoV-2 Droplet Cas12a Assay Enables DNA Quantification from Unamplified 566 Samples at the Single-Molecule Level Acknowledgments 500 We would like to thank HKUST NFF for microfluidic chip fabrication, and HKUST BioCRF for 501 the essential equipment supports. This work is supported by the University-Industry Collaboration is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint Competing interests 513 The authors declare no competing interests. 514 515 It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 24, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted August 24, 2021. ; https://doi.org/10.1101/2021.08.18.21262201 doi: medRxiv preprint