\n<\/p>\n
Acidaminococcus<\/i> sp. Cas12a (AsCas12a) and Lachnospiraceae<\/i> bacterium Cas12a (LbCas12a) were from Tolo Biotech. AsCas12a Ultra was from Integrated DNA Technologies, Inc. EnGen LbaCas12a and NEbuffer r2.1 were from New England Biolabs. The RNA sequences and ssDNA sequences (Supplementary Table 1) were from GENEWIZ. HEPES buffer, magnesium chloride, Lipofectamine 3000 and UltraPure diethyl pyrocarbonate (DEPC)-treated H2<\/sub>O were from Thermo Fisher Scientific. TwistAmp basic kit was from TwistDx. DNA amplification powder kit and RNA-amplification powder kit were from Amp-future. Ribonucleotide solution mix, deoxynucleotide solution mix, RNase inhibitor and T7 RNA polymerase were from New England Biolabs. TIANamp Virus DNA\/RNA Kit was from Tiangen. PCR amplification premix kit was from Takara, MolPure Flash Cell\/Tissue Total RNA Kit and RT\u2013qPCR mix Hifair Advanced One Step RT\u2013qPCR SYBR Green Kit were from Yeasen Biotechnology. RNA sequencing was performed by GENEWIZ. BeyoBlue Plus Coomassie Blue Super-Fast Staining Solution was from Beyotime. HEK293T (IM-H222) cells were from Xiamen Immocell Biotechnology Co., Ltd.<\/p>\n Unless otherwise stated, cleavage reactions contained 5\u2032-FAM-labeled target RNA or label-free target RNA (100\u2009nM), 200\u2009nM Cas12a and 400\u2009nM crDNA. The Cas12a\u2013crDNA complex was first prepared, incubated for 5\u2009min at room temperature before being mixed with target RNA, incubated at 37\u2009\u00b0C for 60\u2009min then resolved by 5% native PAGE (37.5: 1) at 4\u2009\u00b0C (0.25\u00d7 TBE buffer), 250\u2009V for 12\u2009min. RNA was visualized using a Bio-Rad gel imaging system.<\/p>\n A hydrolysis ladder (OH\u2212<\/sup>) was obtained by incubating 0.5\u2009\u03bcl 100\u2009\u03bcM 5\u2032-FAM labeled target ssRNA in hydrolysis buffer at 95\u2009\u00b0C for 30\u2009min, before quenching on ice. The control ladder was a synthetic 5\u2032-FAM-labeled 24-nt RNA fragment derived from target ssRNA.<\/p>\n The reaction was quenched by Inactivation\/Precipitation Buffer (Thermo Fisher Scientific) and precipitation by microcentrifuge at 38,200g<\/i> for 15\u2009min at 4\u2009\u00b0C before adding RNA gel-loading buffer (90% glycerol and 10% formamide). All products were resolved by 15% denaturing PAGE.<\/p>\n Unless otherwise stated, to avoid dissociation of the Cas12a\u2013crDNA or Cas12a\u2013crRNA, complex at low concentrations during target ssRNA binding experiments, binding reactions contained a constant excess ratio of Cas protein and crDNA\/crRNA\/gRNA (2:1), and 50\u2009nM of 5\u2032-FAM-labeled target ssRNA. Reactions were incubated at 37\u2009\u00b0C for 60\u2009min before being resolved by 6% native PAGE (37.5:1) at 4\u2009\u00b0C (0.25\u00d7 TBE buffer), 120\u2009V for 60\u2009min. RNA and DNA were visualized by Bio-Rad gel imaging system.<\/p>\n For ternary complex analysis, binding reactions contained constant 5\u2032-FAM-labeled target RNA (50\u2009nM), then preparation of 200\u2009nM Cas12a\u2013crDNA, or Cas12a\u2013crRNA complex (at a ratio of 2:1) and incubating for 30\u2009min at room temperature, then gradient dilution to target concentration.<\/p>\n For binary complex analysis, binding reactions contained constant crDNA (50\u2009nM), and increasing concentrations of Cas12a.<\/p>\n Unless otherwise stated, 5\u2032-FAM- and 3\u2032-BHQ1-labeled ssDNA probes were synthesized by GENEWIZ. For the general system, reactions were assembled with 50\u2009nM Cas12a protein, 100\u2009nM crDNA, 0.10\u2009pM\u201320\u2009nM activators and 500\u2009nM probes in a total volume of 20\u2009\u03bcl in PCR tube (Bio-Rad). The reaction was performed at 37\u2009\u00b0C, with the buffer containing 10\u2009mM Tris-HCl (pH\u20097.9), 50\u2009mM NaCl, 10\u2009mM MgCl2<\/sub> and 10\u2009mg\u2009ml\u22121<\/sup> bovine serum albumin (BSA). The fluorescent signal (485\u2009nm excitation and 535\u2009nm emission) was monitored with a qPCR system (Bio-Rad) with an interval time of 1\u2009min.<\/p>\n To measure the trans<\/i>-cleavage kinetics of DNA-guided and RNA-guided Cas12a enzymes, we first prepared 1\u2009\u03bcM solutions of the Cas12a\u2013crDNA or Cas12\u2013crRNA complex. This was achieved by incubating a mixture of 1\u2009\u03bcM synthetic crDNA or crRNA with at least twofold excess of the corresponding Cas12a at 4\u2009\u00b0C for 5\u2009min on an ice plate. Cas12a\u2013crDNA or Cas12\u2013crRNA complexes were then activated for trans<\/i>-cleavage activity by mixing and incubating these complexes with synthetic RNA or dsDNA at 100\u2009nM concentration at 4\u2009\u00b0C for 5\u2009min on an ice plate and then incubating on a heating plate at 37\u2009\u00b0C for 30\u2009min. The latter step yielded a solution with an activated Cas12a concentration of 100\u2009nM. We performed the trans<\/i>-cleavage kinetics assay using 1\u2009nM activated Cas12a and varied ssDNA reporter concentrations of 1,000; 2,000; 4,000; 8,000; 16,000 and 32,000\u2009nM. Two replicates were taken for each concentration. The reaction velocities versus reporter concentration data were fitted to the Michaelis\u2013Menten equation using GraphPad Prism v.10 to obtain the k<\/i>cat<\/sub>.<\/p>\n Limited trypsin proteolysis assays were performed using 2.5\u2009\u00b5g of purified AsCas12a in the absence or presence of crDNA at a 1:1.2 molar ratio. Protein samples were incubated with trypsin at a protease-to-substrate mass ratio of 1:100. Proteolysis reactions were carried out at 37\u2009\u00b0C and quenched after 10\u2009min by the addition of SDS\u2013PAGE loading buffer. Reaction products were resolved by SDS\u2013PAGE on precast 5% polyacrylamide gels and visualized by Coomassie staining using BeyoBlue Plus Coomassie Blue Super-Fast Staining Solution.<\/p>\n Unless otherwise stated, FAM-labeled RNA or DNA oligonucleotides were procured from GENEWIZ. To prepare dsDNA substrates, the oligos were annealed at a concentration of 10\u2009\u00b5M in an annealing buffer composed of 10\u2009mM Tris-HCl (pH\u20097.5) and 100\u2009mM NaCl. The solution was heated at 95\u2009\u00b0C for 5\u2009min and allowed to cool down gradually to room temperature. Once annealed, the dsDNA was diluted to a final concentration of 20\u2009nM using a binding buffer containing 10\u2009mM Tris-HCl (pH\u20097.9), 50\u2009mM NaCl, and 10\u2009mg\u2009ml\u22121<\/sup> BSA.<\/p>\n To form the Cas DNP, or Cas RNP, Cas12a and its respective crDNA or crRNA were mixed at concentrations of 2\u2009\u00b5M and 1\u2009\u00b5M, respectively, in the binding buffer. This mixture was then incubated at room temperature (~25\u2009\u00b0C) for 30\u2009min. The DNP or RNP were subsequently serially diluted using the binding buffer. Equal volumes of the diluted DNP or RNP and target substrates were combined, resulting in a final concentration of 10\u2009nM target. The reactions were incubated at room temperature for an additional 60\u2009min. Fluorescence polarization reading of FAM fluorophore was then taken using the Molecular Devices FlexStation v.3 Multi-mode Microplate Reader (Thermo Fisher Scientific).<\/p>\n Unless otherwise stated, FAM-labeled crDNA or crRNA oligonucleotides were procured from GENEWIZ. Cas12a was serially diluted using the binding buffer containing 10\u2009mM Tris-HCl (pH\u20097.9), 50\u2009mM NaCl, and 10\u2009mg\u2009ml\u22121<\/sup> BSA. Equal volumes of the diluted Cas12a and crDNA or crRNA were combined, resulting in a final concentration of 10\u2009nM guide nucleic acid. The reactions were allowed to incubate at room temperature for 60\u2009min. Fluorescence polarization reading of FAM fluorophore was then taken using the Molecular Devices FlexStation 3 Multi-mode Microplate Reader (Thermo Fisher Scientific).<\/p>\n In the RPA reaction, a 50\u2009\u03bcl RPA reaction mixture contained 29.5\u2009\u03bcl primer-free rehydration buffer, 1.2\u2009\u03bcl forward primer (20\u2009\u03bcM), 0.75\u2009\u03bcl reverse primer (20\u2009\u03bcM), 5\u2009\u03bcl sample, 2.5\u2009\u03bcl Mg acetate (280\u2009mM) and 11.05\u2009\u03bcl DNase\/RNase-free water. The reaction was then incubated in 37\u2009\u00b0C for 20\u2009min.<\/p>\n In the RT\u2013RPA reaction, a 50\u2009\u03bcl RT\u2013RPA reaction mixture contained 29.5\u2009\u03bcl primer-free rehydration buffer, 1.2\u2009\u03bcl forward primer (20\u2009\u03bcM), 0.75\u2009\u03bcl reverse primer (20\u2009\u03bcM), 5\u2009\u03bcl sample, 2.5\u2009\u03bcl Mg acetate (280\u2009mM), 1\u2009\u03bcl Script IV RT (200\u2009U\u2009\u03bcl\u22121<\/sup>) and 10.05\u2009\u03bcl DNase\/RNase-free water.<\/p>\n An optimized 20-\u00b5l DNA-guided CRISPR\u2013Cas12a reaction was formulated, consisting of 50\u2009nM Cas12a, 500\u2009nM crDNA, 500\u2009nM fluorescence reporter, 1\u00d7 NEB r2.1 buffer and 2\u2009\u00b5l of amplification solution placed on the lid. The amplification reaction also included 0.125\u2009U\u2009\u00b5l\u22121<\/sup> T7 RNA polymerase, 1\u00d7 T7 RNA polymerase buffer, 0.25\u2009mM rNTP, 0.48\u2009\u00b5M primers and 1.6\u2009\u00b5l of RPA or RT\u2013RPA rehydration mixture (RPA kit from Amp-Future). The tube lid was incubated at 40\u2009\u00b0C for 15\u2009min to promote the production of RNA amplicons. Subsequently, 2\u2009\u00b5l of the amplification solution was spun down and incubated at 37\u2009\u00b0C for 30\u2009min to allow the DNA-guided CRISPR\u2013Cas12a reaction to proceed.<\/p>\n SARS-CoV-2 raw samples were obtained and inactivated in the Prince of Wales Hospital (Hong Kong). Clinical samples were pretreated for pure RNA collection using a TIANamp Virus DNA\/RNA Kit (Tiangen; Beijing; China) and a fast column extraction kit within 20\u2009mins.<\/p>\n First, a 10\u2009\u03bcl reaction mixture contained 0.1\u2009\u03bcl Script IV reverse transcriptase enzyme, 0.5\u2009\u03bcl reverse primer (10\u2009\u03bcM), 1\u2009\u03bcl dNTP mix (10\u2009\u03bcM), 5\u2009\u03bcl RNA template and 3.4\u2009\u03bcl DEPC-treated H2<\/sub>O was incubated for 10\u2009mins at 50 \u00b0C. Notably, the RNA template-primer mix was preheated at 65 \u00b0C for 5\u2009mins, and then incubated on ice for at least 1\u2009min for annealing before reaction. Second, SARS-CoV-2 raw samples were validated with Takara Taq premix kit. In the qPCR reaction, a 25\u2009\u03bcl reaction mixture contained 12.5\u2009\u03bcl premix Taq, 0.5\u2009\u03bcl forward primer (10\u2009\u03bcM), 0.5\u2009\u03bcl reverse primer (10\u2009\u03bcM), 1\u2009\u03bcl probe (10\u2009\u03bcM), 2\u2009\u03bcl cDNA template and 8.5\u2009\u03bcl DEPC-treated H2<\/sub>O. The qPCR program was 95 \u00b0C for 30\u2009s, then 95 \u00b0C for 5\u2009s, 60 \u00b0C for 20\u2009s, 72 \u00b0C for 10\u2009s for 40 cycles.<\/p>\n Each SLEUTH Cas12a cleavage reaction was added to a 100\u2009\u03bcl tube and reacted in Bio-Rad PCR machine. Fluorescence measurements (FAM channel for SYBR Green based melting curve analysis or HEX channel for trans<\/i>-cleavage signal) were then obtained every 60\u2009s for a period of 1\u2009h using the CFX Opus real-time PCR machine (Bio-Rad). The fluorescence signal datapoints were exported directly from the machine system that are background-subtracted signal treated signal. The s.e.m. (s.d.) of duplicates\/triplicates are shown as error bars.<\/p>\n For mammalian expression, AsCas12a and mCherry were cloned into a pcDNA3.1 vector and linked by a P2A peptide to enable coexpression from a single transcript. EGFP was cloned separately into a pcDNA3.1 vector. Plasmid synthesis and assembly were carried out by GENEWIZ. Full construct sequences are provided in Supplementary Table 1.<\/p>\n Complete Dulbecco\u2019s Modified Eagle Medium (DMEM) was prepared by combining 445\u2009ml of basal DMEM with 50\u2009ml of fetal bovine serum and 5\u2009ml of penicillin\u2013streptomycin in a sterile mixing vessel. The mixture was inverted gently until homogeneous, then stored at 2\u20138\u2009\u00b0C for up to 3\u2009months. To prepare 1\u00d7 phosphate-buffered saline (PBS), 5\u2009ml of 10\u00d7 PBS stock solution was diluted with 45\u2009ml of ultrapure water in a sterile container, mixed by gentle inversion and stored at room temperature for up to 6\u2009months.<\/p>\n HEK293T cells were maintained in complete DMEM and passaged every 2\u20133\u2009days at a 1:3 split ratio. Confluent cultures in 6\u2009cm dishes (approximately 5\u2009\u00d7\u2009106<\/sup> cells per dish) were monitored by phase-contrast microscopy for normal morphology and growth. For subculturing, approximately 30% of the total cell population (\u22481.5\u2009\u00d7\u2009106<\/sup> cells) was harvested by aspirating medium, trypsinizing for 2\u2009min at 37\u2009\u00b0C, and neutralizing with twice the volume of complete DMEM. Cells were collected by centrifugation at 90g<\/i> for 3\u2009min, the supernatant was removed, and the pellet resuspended gently in 1\u2009ml of complete DMEM. The cell suspension was then transferred into a fresh 6\u2009cm dish containing 5\u2009ml of prewarmed medium and incubated at 37\u2009\u00b0C with 5% CO2<\/sub>.<\/p>\n For EGFP reporter system, 1\u2009\u00d7\u2009105<\/sup> cells per well were seeded in 48-well plates 24\u2009h before transfection. Then, 200\u2009ng of EGFP reporter plasmid, 400\u2009ng of AsCas12a-mCherry or 0.3\u2009\u00b5l of crDNA (100\u2009\u00b5M) were mixed with 0.45\u2009\u00b5l of Lipofectamine 3000, 0.6 \u00b5l P3000 (except crDNA) and 30\u2009\u00b5l of Opti-MEM Reduced Serum Medium, respectively. Complexes were allowed to form for 15\u2009min and then added dropwise to each well. Transfected cells were harvested 12\u2009h post-transfection for flow cytometry, fluorescence microscopy observation and qPCR analysis.<\/p>\n For endogenous gene knockdown, 1\u2009\u00d7\u2009105<\/sup> cells per well were seeded in 48-well plates 24\u2009h before transfection. Then, 400\u2009ng of AsCas12a-mCherry or 0.3\u2009\u00b5l of crDNA (100\u2009\u00b5M) were mixed with 0.45\u2009\u00b5l of Lipofectamine 3000, 0.6\u2009\u00b5l P3000 (except crDNA) and 30\u2009\u00b5l of Opti-MEM Reduced Serum Medium, respectively. Complexes were allowed to form for 15\u2009min and then added dropwise to each well. Transfected cells were harvested 12\u2009h post-transfection for qPCR and transcriptome sequencing analysis.<\/p>\n At 12\u2009h after transfection as described above, cells were trypsinized with 1\u00d7 Trypsin-EDTA. Then the cells were resuspended in cytometry buffer (0.6% BSA, 2\u2009mM EDTA, 10\u2009mM HEPES, pH\u20097.4 in 1\u00d7 DPBS). Each sample was passed through a 35-\u00b5m cell strainer. Cells were then analyzed in a BD FACSDiscover S8 Cell Sorter. FCS files were then analyzed in FloJo v.10.10 to obtain the MFI of EGFP fluorescence in mCherry\u2013cy5-positive cells.<\/p>\n Total RNA was extracted from samples using the MolPure Flash Cell\/Tissue Total RNA Kit (Yeasen Biotech) according to the manufacturer\u2019s instructions. Purified RNA was subsequently subjected to one-step RT\u2013qPCR using the Hifair Advanced One Step RT\u2013qPCR SYBR Green Kit. RT\u2013qPCR was performed under the following cycling conditions: reverse transcription at 50\u2009\u00b0C for 20\u2009min, initial denaturation at 95\u2009\u00b0C for 5\u2009min, followed by 40 cycles of denaturation at 95\u2009\u00b0C for 15\u2009s and annealing\/extension at 60\u2009\u00b0C for 30\u2009s. Fluorescence signals were acquired using a Bio-Rad real-time PCR system. Relative gene expression levels were quantified using the comparative Ct (2\u2212\u0394\u0394Ct<\/sup>) method, with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH<\/i>) gene serving as the internal reference transcript.<\/p>\n The initial crystal structures for Acidaminococcus<\/i> sp. Cas12a (AsCas12a) were retrieved from the Protein Data Bank (PDB) under accession codes 4UN3 and 5B43, respectively.<\/p>\n To predict the 3D structure of our crDNA-guided RNA targeting tri-complex with cas12a, we used the following computational workflow: first, the primary sequences of the target DNA, guide RNA and the cas12a protein were submitted to the AlphaFold server for structure prediction. The resulting protein\u2013nucleic acid complex model was then optimized and prepared for molecular dynamics simulation using the CHARMM-GUI webserver, generating a GROMACS-compatible topology. The system underwent energy minimization followed by equilibration under constant number of particles, volume and temperature (NVT ensemble) and constant number of particles, pressure and temperature (NPT ensemble). Finally, a 300-ns molecular dynamics simulation was performed to allow structural relaxation and to refine the binding interactions within the predicted complex. Similar workflow was adopted for PAM-related mutation with AsCas12a as a binary complex prediction.<\/p>\n AsCas12a Ultra (6.4\u2009\u00b5M) was incubated with crDNA (final 13\u2009\u00b5M) and RNA (final 13\u2009\u00b5M) in 10\u2009mM Tris-HCl pH\u20098.0 and 50\u2009mM NaCl at 37\u2009\u00b0C for 30\u2009min to form the ternary complex. Then, 4\u2009\u03bcl of 2.5-fold diluted ternary complex was then applied to holey carbon grids (C-flat R1.2\/1.3 Au, 300 mesh) after glow discharge (15\u2009mA, 45\u2009s). After incubation for 10\u2009s at 4\u2009\u00b0C and 100% humidity, the grids were blotted for 4\u2009s with blotting paper, and then plunged quickly into liquid ethane cooled by liquid nitrogen with a Mark IV Vitrobot (Thermo Fisher Scientific). The grids were loaded into a FEI Titan Krios G3i electron microscope (Thermo Fisher Scientific) equipped with a high-brightness field emission gun operated at 300\u2009kV. Images were collected automatically with a K3 Summit direct electron detector (Gatan) using EPU software (Thermo Fisher Scientific) in counting mode and magnification of \u00d781,000, corresponding to a 1.05\u2009\u00c5 physical pixel size. The slit width of the Gatan Imaging Filter (GIF) Bio Quantum was set to 20\u2009eV. The defocus ranges, dose rate and other parameters during image acquisition of the dataset are listed in Supplementary Table 4.<\/p>\n Drift correction and CTF correction were performed in CryoSPARC v.4.7.155<\/sup>. Motion-corrected sums with dose-weighting were used for all subsequent image processing. Blob picking on 500 micrographs was first used to obtain the initial particle stack. After cleaning with two-dimensional classification and heterogenous refinement, the particles of classes with clear AsCas12a features were used as templates to pick on the full 2,030 micrographs. Two rounds of heterogenous refinements were then used to obtain a clean stack of 1,305,848 particles, which showed preferred orientation. Rebalance orientations in CryoSPARC v.4.7.156<\/sup> was used twice to remove excessive side views, resulting in 277,781 particles as the final stack to reconstruct the consensuses map for subsequent 3D classifications in CryoSPARC v.4.7.155<\/sup> and Relion v.556<\/sup>. The data processing flowchart is shown in Supplementary Fig. 9.<\/p>\n Models of PDB 8SFL were first docked into the final map using fit in map function of ChimeraX v.1.957<\/sup>. The models of bound crDNA and RNA were then fitted into the density, matching the distinct purine\/pyrimidine patterns and their known sequences. Coot v.0.9.8.9658<\/sup> was used to adjust the model manually. The model was refined against the corresponding cryo-EM density map with phenix.real_space_refine module in PHENIX v.1.21.2-5419 package59<\/sup>. The qualities of the final refined model were estimated with MolProbity60<\/sup>. The figures on maps and refined models were generated with ChimeraX v.1.9. Refinement statistics are presented in Supplementary Table 4.<\/p>\n Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.<\/p>\n<\/div>\n\n","protected":false},"excerpt":{"rendered":" Materials Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a) were from Tolo Biotech. AsCas12a Ultra was from Integrated DNA Technologies, Inc. EnGen LbaCas12a and NEbuffer...<\/p>\n","protected":false},"author":1,"featured_media":365,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"fifu_image_url":"https:\/\/media.springernature.com\/m685\/springer-static\/image\/art%3A10.1038%2Fs41587-026-03120-5\/MediaObjects\/41587_2026_3120_Fig1_HTML.png","fifu_image_alt":"","footnotes":""},"categories":[1],"tags":[431,433,422,432,430,429,428,423,427,424,425,426],"class_list":["post-364","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-rj","tag-agriculture","tag-bioinformatics","tag-biomedical-engineering","tag-biomedical-engineering-biotechnology","tag-biomedicine","tag-biotechnology","tag-general","tag-infectious-disease-diagnostics","tag-life-sciences","tag-molecular-biology","tag-nucleic-acid-therapeutics","tag-synthetic-biology"],"_links":{"self":[{"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/posts\/364","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=364"}],"version-history":[{"count":0,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/posts\/364\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=\/wp\/v2\/media\/365"}],"wp:attachment":[{"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=364"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=364"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/rjbarrett.redirectme.net\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=364"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}RNA cleavage assays<\/h3>\n
RNA cleavage site mapping<\/h3>\n
Electrophoretic mobility shift assays<\/h3>\n
\n Trans<\/i>-cleavage activity analysis<\/h3>\n
Michaelis\u2013Menten modeling<\/h3>\n
Limited proteolysis<\/h3>\n
ssRNA, ssDNA or dsDNA target binding K<\/i>
\n d<\/sub>
\n 2<\/sub> measurement using fluorescence polarization<\/h3>\ncrDNA or crRNA binding K<\/i>
\n d<\/sub>
\n 1<\/sub> measurement using fluorescence polarization<\/h3>\nRecombinase polymerase amplification<\/h3>\n
SLEUTH detection<\/h3>\n
SARS-CoV-2 clinical sample collection<\/h3>\n
SARS-CoV-2 detection with RT\u2013qPCR<\/h3>\n
Analysis of SLEUTH fluorescence data<\/h3>\n
Plasmid construction<\/h3>\n
RNA silencing reagent preparation<\/h3>\n
Cell culture<\/h3>\n
Cell transfection<\/h3>\n
Flow cytometry for quantification of EGFP expression<\/h3>\n
RT\u2013qPCR for relative quantification of mRNA<\/h3>\n
Structure prediction pipeline using AlphaFold3-guided molecular dynamic simulation<\/h3>\n
Cryo grid preparation and data collection<\/h3>\n
Model building<\/h3>\n
Reporting summary<\/h3>\n