Methods

A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

Constructs and reagents

The coding regions of LwaCas13a (Leptotrichia wadeii Cas13a)56, AsCas12a (Acidaminococcus sp. Cas12a)57 and LbCas12a (Lachnospiraceae bacterium Cas12a)57 were inserted into pET28a expression vector between BamHI and XhoI restriction enzyme sites for prokaryotic expression and purification of these proteins. N gene fragments (two different regions: #1 and #2) from SARS-CoV-2 viral genome were inserted into pHAGE-EF1α-puro vector between BamHI and KpnI restriction enzyme sites for SARS-CoV-2 pseudovirus detection. Oligonucleotides used in this study (Supplementary Table 2) were synthesized from HuaGene Biotech (Shanghai, China), Synbio Technologies (Suzhou, China) and GENEWIZ (Suzhou, China). Detailed information about reagents and instruments, including the commercial vendors and item numbers, is provided in Supplementary Table 3.

Nucleic acid preparation

For preparation of DNA templates of SARS-CoV-2 ORF1ab, N and S genes, PCR amplification was performed by indicated primers with the forward primer containing an appended T7 promoter sequence using the template prepared through annealing of two synthetic oligonucleotides (Supplementary Table 2). For preparation of RNA templates, the in vitro transcription was performed with T7 promoter-inclusive DNA templates. Briefly, the in vitro transcription (IVT) system consisted of 4 μL 10x Transcription Buffer, 3.125 mM rNTPs, 50U T7 RNA Polymerase (Lucigen), 1 μL RNase Inhibitor and 50~100 ng DNA template, and the total reaction volume was 40 μL. After thorough mixing, the reaction system was incubated at 37°C for 1~2 hour (h). For preparation of crRNAs, the DNA oligonucleotide containing reverse complementary sequence of crRNA was annealed to an oligo (T7-F) with T7 promoter sequence to form a partial duplex DNA template for IVT. Then crRNAs were in vitro transcribed using the same IVT reaction system as described above. RNA templates and crRNAs were purified by RNA Clean & Concentrator-5 Kit (Zymo research), and quantified by the high sensitivity RNA Qubit fluorometer (Thermo Fisher).

Cas protein expression and purification

Cas protein expression plasmids (pET28a-AsCas12a, pET28a-LbCas12a and pET28a-LwaCas13a) were transformed into Escherichia coli. Rosseta™ 2(DE3)pLySs competent cells. After transformation, cells were plated on kanamycin and chloramphenicol positive Luria-Bertani (LB) agar plate, and incubated for 16 h at 37°C. Pick up one colony from the plate, inoculate in 5 mL liquid LB medium supplemented with kanamycin and chloramphenicol, and put the starter culture on a shaker at 37°C overnight. 5 mL of starter culture was used to inoculate 1L of LB media supplemented with antibiotics and shaked at 37°C with 300 r.p.m. Cultures were allowed to grow until OD600 reached 0.4~0.6, and then cooled down for 30 minute (min) at 4°C. Add isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM to induce protein expression for 14-16 h at 300 r.p.m. in a pre-chilled 20°C shaker. After induction, the cells were harvested by centrifugation (5000 g, 4°C, 10 min) for later purification and stored at −80°C.

Protein purification procedures were performed at 4°C. Cell pellet was resuspended in 15 mL of lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM DTT, 5% glycerol, 1 mM PMSF, 1 mg/mL lysozyme). The cell lysate was then sonicated by the sonicator with the following parameters: sonication (φ6, power 40%) for 1 second on and 2 seconds off with a total sonication time of 15 min. The sonicated sample was then centrifuged at 14000 r.p.m. for 10 min at 4°C and the supernatant was mixed with an equal volume of equilibration/wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole; pH 7.4). Since the recombinant Cas protein contains His-tag, HisPur™ Cobalt Resin (Thermo Fisher, #89964) was utilized to pull-down the protein. Washed protein extract was mixed with the prepared cobalt resin on end-over-end rotator for 30 min at 4°C followed by washing of the protein-bound cobalt resin twice in equilibration/wash buffer. Elute bound His tagged protein using elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole; pH 7.4) twice. Zeba™ Spin Desalting Columns (Thermo Fisher, #89890) was used to desalt protein, and ~2 mL protein could be collected after centrifuging at 850 g for 2 min. Mix the protein with 10 mL Storage Buffer (600 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5% glycerol, 2 mM DTT), and transfer the mix into Amicon® Ultra-15 Centrifugal Filter Devices (Millipore, #UFC905008) to concentrate the protein and exchange the storage buffer as well. The concentration of purified proteins was quantified by BCA protein assay kit (Meilunbio, #MA0082). SDS-PAGE analysis was performed with samples collected after different purification steps and the results were visualized by Coomassie Blue staining. Purified protein was stored at −80°C as 10 μL aliquots at a concentration of 2 mg/mL.

CRISPR detection assay

CRISPR-LwaCas13a system was used for RNA detection. The standard LwaCas13a-based detection assay was performed at 37°C with 1x CutSmart buffer, 100 nM LwaCas13a protein, 100 nM crRNA, 250 nM RNA reporter and 1 μL of nucleic acid target in a 20 μL reaction system. For DNA detection system, CRISPR-AsCas12a and CRISPR-LbCas12a systems were applied. The standard reaction systems of Cas12a-based nucleic acid detection consisted of 1x CutSmart buffer, 50 nM AsCas12a protein or LbCas12a protein, 50 nM crRNA, 250 nM DNA reporter, and 1 μL of nucleic acid target or amplification products in a final volume of 20 μL at 37°C for 1 h. For the optimization of the detection systems, indicated amount of components and specified chemicals were added to the detection systems.

Fluorescence readout

The CRISPR-LwaCas13a and CRISPR-AsCas12a (LbCas12a) fluorescence detection assays were performed by using fluorophore-quencher (FQ) reporters involving a short single-stranded (ss) RNA or DNA oligonucleotide, respectively. Both of the ssRNA and ssDNA FQ reporters were composed of a 6-FAM fluorophore on 5 terminal and a BHQ1 quencher on 3 terminal (ssRNA FQ reporter: 5’ -/6-FAM/UUUUUU/BHQ1/-3’; ssDNA FQ reporter: 5’ -/6-FAM/TTATT/BHQ1/-3’). For LwaCas13a-based assay, the detection system consisted of 100 nM LwaCas13a, 100 nM crRNA, 250 mM ssRNA fluorescent reporter, 1x CutSmart Buffer and 1 μL nucleic acid target in a 20 μL reaction system. For AsCas12a- and LbCas12a-based assays, the detection system contained 50 nM AsCas12a or LbCas12a protein, 50 nM crRNA, 250 nM ssDNA fluorescence reporter, 1x CutSmart Buffer and 1 μL DNA target or amplification products. Fluorescence signal was dynamically measured by QuantStudio™ 5 Real-Time PCR System (Thermo Fisher). Background-subtracted signals for each monitoring points were further normalized by subtraction of its initial value to make comparison between different conditions (arbitrary unit, a.u.) for the analysis. Visual detection was accomplished by imaging the tubes through E-Gel™ Safe Imager™ Real-Time Transilluminator (Thermo Fisher).

Lateral flow readout

For lateral flow strip assays, the AsCas12a-based detection system was assembled as described above except that the fluorescence reporter was replaced with the biotin-labeled reporter. Lateral flow cleavage reporter (5’ -/6-FAM/TTATTATT/Biotin/-3’) was added to the reaction at a final concentration of 250 nM in 20 μL reaction volume along with the 1 μL RT(reverse transcription)-LAMP product, and incubate the reaction at 37°C for 1 h. After completion of the incubation, add 80 μL HybriDetect Assay Buffer into the reaction tube. A lateral flow strip (Milenia Biotec GmbH, # MGHD 1) was placed to the reaction tube and incubated for approximately 3~5 min, and the result was visualized directly. The negative result is indicated by one primary band at the control line (C), and significant band in test line (T) represents positive result.

LAMP assay

For detection of SARS-CoV-2 RNA, RT-LAMP reactions were performed with caution on a dedicated clean bench using filter tips to prevent sample contamination. One-step RT-LAMP mix was assembled with 1 μL 10x isothermal amplification buffer, 1.4 mM of dNTPs, 6.5 mM MgSO4, 3.2 U Bst 2.0 (NEB, #M0538S), 3 U WarmStart RTx Reverse Transcriptase (NEB, #M0380S), 0.2 μM F3/B3 primers, 1.6 μM FIP/BIP primers, 0.8 μM LF/LB primers, and 1 μL of RNA template in a 10 μL volume, and then incubated at 62°C for 15~30 min. Primers for LAMP are listed in Supplementary Table S2. The heat inactivation (80°C for 20 min) of LAMP product was performed to reduce the background signal before proceeding to CRISPR detection assays. Quantitative real-time PCR was conducted using non-specific DNA-binding dye EvaGreen (Biotium, #31000) to quantitate the total amount of amplification products. EvaGreen-derived fluorescence signal was normalized by subtraction of initial value to make comparison between different conditions. For evaluation of the target specificity of LAMP reactions, amplification products were purified by PCR Purification Kit (Thermo Fisher, #K0702), and followed by fluorescence detection with AsCas12a system. The purified products might be diluted to an appropriate concentration to make sure the detection signal fell into the effective detection range.

RPA assay

RPA assays were set up using commercial RPA kit (TwistDx). One step RT-RPA reaction system was composed of 9 μL of RPA solution (primer-free rehydration buffer), 224 mM of MgOAc, 40 U of ProtoScript II Reverse Transcriptase (NEB, #M0368S), 0.5 μM of forward primer, 0.5 μM of reverse primer and add UltraPure water to 16 μL. Primers for RPA are listed in Supplementary Table S2. The mixture was incubated at 40°C for 30 min, followed by heat inactivation for 20 min at 80°C. Total amount of amplification product was quantified by quantitative real-time PCR using EvaGreen dye, and specific target amplification was determined by AsCas12a-based CRISPR detection after purification using PCR Purification Kit.

Pseudovirus production and detection

HEK293FT cells were employed to pack the SARS-CoV-2 pseudoviral particles. Cells were cultured with DMEM medium supplemented with 10% fetal bovine serum. DNA Transfection was performed in 6-well plates by Lipofectamine™ 2000 Transfection Reagent with a mix of 1.5 μg pHAGE-EF1α-puro plasmid carrying SARS-CoV-2 N gene fragment (#1 and #2), 0.75 μg pCMVR8.74, and 0.5 μg pMD2.G. The viral particle-containing supernatant was harvested at 48 h post transfection and centrifuged at 3000 r.p.m for 5 min to remove the cell debris. Aliquot and store the virus supernatant at −80°C before use. For virus titration, a commercial Lentivirus Titer Kit was used (Abm, #LV900). Briefly, 1 μL virus supernatant was lysed in 9μL Virus Lysis Buffer for 3 min at room temperature. Quantitative Reverse Transcription PCR (qRT-PCR) was performed with specified primer set to quantify the viral particles. The titer of virus can be calculated from on-line lentiviral titer calculator which is provided by Abm at http://www.abmgood.com/High-Titer-Lentivirus-Calculation.html. To simulate the actual application, viral particles were firstly transferred into the Viral Transport Media (VTM) of Sample Collection Kit (BEAVER, #43903) to a final volume of 1 mL as a mimic of nasopharyngeal swab collected sample. Take out 100 μL solution for RNA extraction by UNIQ-10 Column Trizol Total RNA Isolation kit (Sangon, #B511321-0100), and elute the RNA by 50 μL Ultrapure H2O. RT-LAMP was performed with 1 μL extracted RNA as template followed by AsCas12a-based detection in the absence or presence of L-proline.

Statistical analysis

Statistic significances were calculated by GraphPad Prism 8.4.0 and all the data were shown as mean ±s.d. The two-way ANOVA with Sidak’s multiple comparisons test was used to compare differences between groups. Statistical significance was determined by an unpaired two-tailed t-test. Asterisks indicate **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article TitleA Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

Abstract

The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.


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