Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.
Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.
CRISPR-based diagnostics for the detection of nucleic acid biomarkers was first reported in 20171,2,3,4, and since then, has been proven as next-generation diagnostics with Food and Drug Administration (FDA)-approved tests, particularly for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA, in multiple countries5,6,7,8. Beyond coronavirus disease 2019 (COVID-19), the technologies have been demonstrated as effective for detecting diverse viruses and bacteria9,10,11,12,13, genetic disease mutations and deletions, and can be engineered to detect protein and small molecule biomarkers2,12. CRISPR-based diagnostics often combine isothermal amplification methods-such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP)14,15-with CRISPR-based detection using RNA-guided CRISPR-associated (Cas) enzymes with "collateral" endonuclease activity after target recognition3. The dual use of isothermal amplification and CRISPR-based detection confers several desirable attributes to the technologies, particularly high diagnostic accuracy approaching that of the gold-standard polymerase chain reaction (PCR), and capability to multiplex and detect small sequence differences, including single-nucleotide polymorphisms1,9.
The most accurate versions of CRISPR-based diagnostics, however, contain multiple components and steps and can be complicated to perform with non-experts or at the point of care (POC). To address this challenge of extending the use of highly accurate CRISPR-based diagnostics to POC settings, we have developed protocols to prepare premixed, lyophilized, recombinase-based isothermal amplification and CRISPR-based detection reactions, which are easy to use and store7. These protocols should complement existing excellent protocols on CRISPR-based diagnostics14, which contain additional information on the production of biochemical components needed for CRISPR-based diagnostics7, design guidelines1, and formulations using alternative isothermal amplification techniques2,14,15,16 and Cas enzymes12. Ultimately, we hope that CRISPR-based diagnostics can help realize rapid, inexpensive, and sensitive nucleic detection in settings where portable and instrumental-free analyses are required1,9,17.
We primarily use the combination of RPA and Cas13-based detection in our protocols. RPA functions at near-ambient temperatures (37-42 °C), and therefore, has low energy and equipment requirements. Other isothermal amplification reactions require higher temperatures (LAMP, 60-65 °C; strand-displacement amplification (SDA), 60 °C; exponential amplification reaction (EXPAR), 55 °C; and helicase-dependent amplification (HDA), 65 °C)2. The design of RPA primers is also not complex, unlike LAMP primers, and can be extended to multiplexed amplification7,9,14. Even though multiplexing beyond two targets with RPA is difficult in practice, there are clear guidelines on how to design multiplexed RPA primers to minimize interference18.
While carryover contamination remains a big issue in the two-step workflow, the generated amplicons of RPA are small in size and less likely to cause carryover contamination compared to large concatemeric amplicons from LAMP14. RPA primers can be designed according to standard guidelines14: they are generally 25-35 nucleotide-long with melting temperatures of 54 to 67 °C. The amplicon size should be smaller than 200 base pairs. The reverse transcriptase enzyme (RT) can be added to RPA to enable amplification from RNA, and in reverse transcription-RPA (RT-RPA), another enzyme Ribonuclease H (RNase H) is typically added in small amounts to help resolve the resulting RNA: DNA hybrid and promote the amplification reaction5,19. To allow T7 transcription for Cas13-based detection, the T7 RNA polymerase promoter can be placed at the 5' end of the forward RPA primer.
After amplicons are generated from RPA, they can be detected with crRNA-programmed Cas enzymes. Among various Cas enzymes that can be used for amplicon detection, we prefer RNA-targeting Cas13 variants due to their high cleavage activity1 and well-characterized polynucleotide cleavage preferences7,9, the latter of which can be used for multiplexed detection. The crRNA sequence for Cas13 is designed to be complementary (reverse complement) to the target site in the produced RNA with spacer and direct repeat (DR) sequences. The crRNA sequence and RPA primers should not overlap, to avoid undesired Cas-based detection of off-target amplification products, which increases background and false positives14.
Cas-based detection relies on different detection modalities to monitor the cleavage of reporter nucleic acid molecules (RNA reporters for Cas13-based reactions)1,2,14. Different detection modalities include colorimetry, electrochemical readouts, fluorescence, sequencing readouts, and lateral flow strips2. We focus on the fluorescence readout given a variety of fluorophores and reporters such as cyanine 5 (Cy5), rhodamine X (ROX), and carboxyfluorescein (FAM), which can be effectively excited using a single blue LED light source, and their fluorescence analyzed by a microplate reader or a real-time thermal cycler7,14. In addition, fluorescence readout is easy to set up and allows continuous monitoring, which enables real-time quantitation. Multiplexed RPA preamplification and multiplexed Cas13-based detection using Cas enzymes can be combined with multicolor fluorescence readouts for the simultaneous detection of multiple genetic targets2,7.
In our protocols, we first isothermally amplify RNA with RT-RPA by adding the RNA sample to the lyophilized form of the RT-RPA reagent (Figure 1). During RT-RPA, the T7 promoter is added to the generated double-stranded DNA amplicon. Thereafter, the RPA products are transferred to the lyophilized Cas13-based detection, which also contains T7 RNA polymerase. This detection reaction will convert DNA amplicons into RNA (via T7 RNA polymerase), which can be readily detected with crRNA-programmed Cas13. Target-activated Cas13 will cleave reporter molecules to produce detectable fluorescence signal2,7,14. While the protocols here pertain to detection by Leptotrichia wadei (LwaCas13a), they can be extended to simultaneous, multiplexed detection of up to four targets using orthogonal Cas13 and Cas12 enzymes7,9. RPA and Cas-based detection reactions can also be combined into a single tube, albeit at a loss of sensitivity14.
Figure 1: CRISPR-based detection workflow. The workflow illustrates the detection of two target genes. First, regions of interest within the DNA target are isothermally amplified with RPA; the reaction can be performed with reverse transcription (RT-RPA) when detecting RNA targets. Thereafter, T7 transcription converts dsDNA amplicons to RNAs, which in turn are recognized by Cas13-crRNA complexes capable of eliciting collateral RNase activity upon target binding. Cleavage of quenched fluorescence reporters produces fluorescence signals that can be monitored using a microplate reader, visualized by an LED transilluminator, or used with a real-time thermal cycler. Abbreviations: CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; RT = reverse transcription; RPA = recombinase polymerase amplification; Cas = CRISPR-associated protein; LED = light-emitting diode. Please click here to view a larger version of this figure.
The key features of the protocols presented here are the premixing formulations for lyophilization. Premixing simplifies the reaction use and enhances reproducibility, but many components within RPA and Cas-based detection-particularly reverse transcription and some labile cofactors such as ATP are not stable in solution. Therefore, we formulate the premixed solutions such that they can be freeze-dried for long-term storage and easy transport and deployment1,9,20. Premixed, freeze-dried reagents also help improve the detection sensitivity, as higher sample volume (and therefore, higher DNA/RNA input) can be added to reconstitute the reaction10. In our formulations, we primarily use trehalose21 as the cryoprotectant in lyophilized RPA and Cas-based detection reactions7. In addition to reagent preservation, trehalose also promotes the reactions via increasing enzyme stabilization16,22,23,24 and decreasing melting temperatures of dsDNA23. Explorations of other stabilizers5,7,21,25,26 beyond trehalose may yield even more optimal formulations for different use scenarios.
1. Preparation of lyophilized RT-RPA premixed reagents
2. Preparation of lyophilized CRISPR-Cas13a premixed detection reagents
NOTE: Follow step 1.1 for equipment preparation.
3. RT-RPA nucleic acid amplification
NOTE: To prevent cross-contamination, workplace areas and pipettors should be separated for pre amplification, sample addition, and post amplification. We recommend using filtered pipette tips.
4. CRISPR-Cas13 nucleic acid detection
We highlight the kinetics of FAM fluorescence signal generation from the combined detection of s and n genes of SARS-CoV-2, and how the information was used to determine optimal conditions for lyophilized, premixed reaction formulations. In all cases, we included samples with Ct values well within the determined detection limit (LoD) (Ct ~31-33), as well as those with Ct at the LoD (Ct ~35-37)7, to allow differentiation of protocols with better sensitivity.
For lyophilized, premixed RT-RPA, we found that the inclusion of KOAc in the premixed reaction negatively affected the reaction sensitivity (Figure 2A). We therefore omitted KOAc from the premixed reaction and added it with the reaction initiator, Mg(OAc)2, once the RNA sample had been added. We also used similar assays to determine the optimal concentration of a cryoprotectant trehalose (Figure 2B) and the effects of triglycine7 in the lyophilized RT-RPA reaction (Figure 2C). Therefore, optimal lyophilized, premixed RT-RPA reactions were prepared with 6% trehalose, no KOAc (but it was added later after the RNA sample), and supplemented with triglycine, as described in protocol section 1.
Figure 2: Optimizing lyophilized, premixed RT-RPA reactions. RT-RPA reaction was prepared using primers targeting the n gene of SAR-CoV-2. Mg(OAc)2 was excluded from all premixed reactions. Effects of (A) KOAc, (B) trehalose, and (C) triglycine added to the premixed reactions were evaluated. Reaction monitoring via generated FAM fluorescence was carried out using a real-time thermal cycler or a fluorescence microplate reader. Individual data from triplicate measurements are shown. Abbreviations: RT = reverse transcription; RPA = recombinase polymerase amplification; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; Cas = CRISPR-associated protein; SAR-CoV-2 = Severe Acute Respiratory Syndrome Coronavirus 2; Ct = cycle threshold; FAM = carboxyfluorescein. Please click here to view a larger version of this figure.
For lyophilized, premixed Cas13a-based reactions, we verified the importance of having the correct cryoprotectant: either 6% trehalose or 2% sucrose19 worked well, while lower sucrose at 1% or 5% w/v PEG20000 did not preserve the reaction efficiency after lyophilization (Figure 3). MgCl2 was omitted from all reactions and was added at the rehydration step. The optimal lyophilized Cas13-based detection reaction was prepared with 6% trehalose as shown in protocol section 2.
Figure 3: Optimizing lyophilized, premixed Cas13-based detection reactions. The detection reaction was performed at 37 °C using the RPA product from the SARS-CoV-2 n gene. The tests were done before and after lyophilization. MgCl2 was excluded from all premixed reactions. The abilities of (A) trehalose, (B) PEG20000, and (C) sucrose in preserving reaction efficiency after lyophilization were evaluated. Reaction monitoring via generated FAM fluorescence was carried out using a real-time thermal cycler. Individual data from triplicate measurements for the "after lyophilization" conditions and one replicate from the "before lyophilization" conditions are shown. Figure 3A,B was taken from Patchsung et al.7. Abbreviations: RPA = recombinase polymerase amplification; SAR-CoV-2 = severe acute respiratory syndrome coronavirus 2; FAM = carboxyfluorescein. Please click here to view a larger version of this figure.
The lyophilized, premixed reagents for RT-RPA and Cas13-based reactions are stable at -20 °C for at least 8 months [Figure 4].
Figure 4: Stability tests of lyophilized, premixed RPA and CRISPR-Cas13a reagents at -20 °C. Stability test at (A) 30 days, (B) 45 days, (C) 3 months, (D) 5 months, (E) 8 months. Individual data from triplicate measurements are shown. Please click here to view a larger version of this figure.
The LoD of lyophilized RT-RPA and Cas13-based reactions for the detection of s and n genes of SARS-CoV-2 is shown in Figure 5. The kinetics of signal generation can be improved upon dual gene detection compared to when only one gene is detected.
Figure 5: Analytical sensitivity of single-gene vs dual-gene detection. (A) The LoD of lyophilized singleplex RT-RPA and Cas-based detection targeting the s gene of SAR-CoV-2. (B) The LoD of lyophilized singleplex RT-RPA and Cas-based detection targeting the n gene of SAR-CoV-2. (C) The LoD of lyophilized multiplex RT-RPA and dual Cas-based detection targeting the s and n genes. The LoD was determined using serially diluted SARS-CoV-2 RNAs, whose Ct values were determined using an RT-qPCR assay targeting the n gene. Individual data from 3-6 replicate measurements are shown. Abbreviations: LoD = limit of detection; RT = reverse transcription; RPA = recombinase polymerase amplification; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; Cas = CRISPR-associated protein; SAR-CoV-2 = Severe Acute Respiratory Syndrome Coronavirus 2; Ct = cycle threshold; qPCR = quantitative polymerase chain reaction. Please click here to view a larger version of this figure.
Beyond virus detection, we showcase a prototype of a POC genotyping tool based on CRISPR diagnostics, to help identify venomous snake species endemic to Thailand (Figure 6). Faster and accurate identification of snake species through DNA samples collected from bite wounds can aid in the prompt administration of antivenoms, improving the patient's chance of survival. We targeted regions within the mitochondrial cytochrome b (cytb) gene of eight snake species including Ophiophagus hannah (King cobra) (Figure 6A) and showed that detection of synthetic DNA surrogates of the O. hannah cytb gene can reach the attomolar level of sensitivity (Figure 6B,C).
Figure 6: A prototype for CRISPR-based detection to identify snake species from snakebite envenoming. (A) RPA primers and LwaCas13a crRNA design for the detection of the cytb gene of Ophiophagus hannah. T7 promotor (T7 overhang) was placed at the 5' end of the forward RPA primer. (B) Kinetics of FAM signal generation over 60 min and (C) endpoint FAM fluorescence from LwaCas13a-mediated detection of synthetic dsDNA inputs corresponding to the cytb gene segments from O. hannah. Different input concentrations (0, 10-9, 10-12, 10-15, and 10-18 M) were used. Data are mean ± s.d. from triplicate measurements. Abbreviations: RPA = recombinase polymerase amplification; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; Cas = CRISPR-associated protein; FAM = carboxyfluorescein. Please click here to view a larger version of this figure.
Lyophilization of CRISPR-Cas13a premixed detection reagents | ||||
Step | Ramp Rate | Shelf Temp | Time (hh:mm) | Vaccuum |
(°C/min) | (°C) | (mbar) | ||
Freeze 1 | 3 | -30 | off | – |
Dry 1 | 3 | -30 | off | 0 |
Dry 2 | 3 | 4 | 01:00 | 0 |
Dry 3 | 3 | 20 | ∞ | 0 |
Table 1: Lyophilizer settings of premixed RT-RPA and CRISPR-Cas13a detection reagents. Abbreviations: RT = reverse transcription; RPA = recombinase polymerase amplification; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; Cas = CRISPR-associated protein.
Parameter | Setting | |
Temperature | 37 °C | |
Kinetic cycle | Duration time | 02:00 (mm:ss) |
Interval time | 02:30 (mm:ss) | |
Shaking | Duration time | 1 sec |
Mode | Linear | |
Amplitude | 1 mm | |
Fluorescence intensity | Excitation wavelength | 488 nm |
Emission wavelength | 520 nm | |
Mode | Top | |
Z-Position | 24470 | |
Gain | 71 | |
Integration time | 20 µs | |
Number of flashes | 25 |
Table 2: Fluorescence microplate reader program for nucleic acid detection.
There are critical steps in this protocol. For area segregation, it is recommended to use separate spaces for nucleic acid extraction, mastermix preparation (pre amplification area), sample addition, and amplicon detection (post amplification area). Each area should be set by using a separate set of tools and equipment. Do not bring tools from one area into another area, especially from the post amplification area into the pre amplification area. Cleaning of working areas is necessary: one should clean working areas, pipettes, vortexers, and permanent markers with RNase/DNase decontamination solution/3% Clorox followed by 70% ethanol or autoclaved deionized water. Washing basic labware and removable parts of instruments, such as 1.5 mL tube plastic racks and the rotor adaptor of a minicentrifuge is required. Nuclease-free water is used as a negative control and a reaction component, and thus is a sensitive reagent that can be contaminated if not handled carefully. So, we recommend making small aliquots of water to be only used once.
Always run a negative control reaction using the assay primers to check for contamination. Additionally, to avoid contamination problems, we recommend preparing the negative control reaction by adding nuclease-free water in the pre amplification area instead of the sample addition room. Magnesium acetate-potassium acetate mixture should be aliquoted in small volumes (~20 µL/tube) that are only used once to avoid reagent contamination with carryover amplicons. When adding the magnesium-potassium acetate mixture to lyophilized premix RPA reactions, we recommend changing the pipette tip every time. For the workflow of experiments, keep the workflow unidirectional, from pre amplification to post amplification experiments. Move from "cleaner" (pre amplification) workspaces toward "dirtier" (post amplification) ones.
For modifications and troubleshooting of the method to avoid carryover contamination of amplicons, we recommend spatially separating the three main steps of the experiment: preparation of the RPA reaction; sample addition; and the amplification and detection steps. The mastermix should be prepared in a template-free area of the lab. When changing rooms, also change gloves and lab coats. Work unidirectionally from pre to post amplification. Avoid using the same pipette for different steps. It is best if different sets of pipettes are arranged for these steps: preparation of mastermixes; addition of the template DNA/RNA; and the transfer of the amplified product. Use filtered tips, try to prevent aerosol formation as much as possible, and avoid touching the inside of the tube when opening it. If amplification experiments show contamination, all reagents should be changed. If possible, go to another lab with fresh reagents and check if contamination persists. Aside from reagents, do not bring anything else including pipettes. Try autoclaving pipettes if they are autoclavable.
To improve the amplification rate (assay optimization), first, consult the manufacturer's instructions such as the RPA manual from TwistDx to ensure optimal designs. Next, try empirical optimizations of the reactions such as adding reaction additives known to boost RPA and CRISPR reactions7; increasing the reaction times (e.g., RPA reaction time up to 60 min); or varying primer sequences and concentrations. We also recommend adding as much sample volume as possible; using lyophilized reagents allows more sample volume to be added. To reduce background fluorescence, one can do a heat inactivation step (75 °C for 5 min) after RPA is finished, before moving on to detection, or optimize the concentration of the reporter. Too much reporter will give a higher background. In addition, adding RNase inhibitors helps to prevent Cas-independent degradation of the reporter.
Beyond optimizing (RT)-RPA and CRISPR-based reaction conditions and their handling procedures, one should ensure that reagents used are of good quality, and that the nucleic acid extraction step preceding all reactions is successful. Lyophilized reagents provide high detection sensitivity, are portable, enable POC testing, and can be applied to diverse clinical samples. Moreover, lyophilized (RT)-RPA can be rehydrated instantly by adding the nucleic acid extract from clinical samples. A previous study showed higher sensitivity when putting a greater volume of nucleic acid input with no restricted volume, effectively increasing the total amount of target nucleic acid.
CRISPR-based diagnostics have been adapted to detect pathogens by collecting clinical samples such as nasopharyngeal and throat swabs, urine, and serum7,8,11. It has also been applied to distinguish single nucleotide variants, identify antibiotic-resistance genes in antimicrobial-resistant organisms, and detect cancer-associated mutations. The utility of the technology may be the greatest in field applications where engineered biochemical reactions for isothermal amplification and CRISPR-based detection can be combined with field-ready devices and readouts such as a handheld fluorometer or lateral flow strips.
The authors have nothing to disclose.
C.U. acknowledges funding from Siam Commercial Bank under VISTEC-Siriraj Frontier Research Center, and from Thailand Science Research and Innovation (TSRI), fundamental fund, fiscal year 2024, grant number FRB670026/0457. R.K. and M.P. are supported by studentship and research assistantship funds from VISTEC, respectively.
Material | |||
Betaine solution | Sigma-Aldrich | B0300-1VL | |
DEPC-Treated water | Invitrogen | AM9915G | |
Dithiothreitol (DTT) | Merck | 3870-25GM | |
EpiScript reverse transcriptase | Lucigen | ERT12925K | |
Gly-Gly-Gly | Sigma-Aldrich | SIA-50239-1G | |
LwaCas13a | Producing in-house | Strain name:Leptotrichia wadei, Abbreviation: Lwa, Protein name: LwaCas13a | |
Magnesium chloride solution, 1M | Sigma-Aldrich | M1028-10X1ML | |
NxGenT7 RNA polymerase | Lucigen | 30223-2 | |
Poly(ethylene glycol) | Sigma-Aldrich | 81300-1KG | |
Potassium acetate solution, 5M | Sigma-Aldrich | 95843-100ML-F | |
Riobnucleotide Solution Mix | NEB | N0466L | |
RNase H | NEB | M0297L | |
Sucrose | TCI | TCI-S0111-500G | |
Trehalose Dihydrate | Sigma-Aldrich | SIA-90210-50G | |
Trizma hydrochloride solution, 1M, pH 7.4 | Sigma-Aldrich | T2194-100ML | |
TwistAmp Basic kit | TwistDx | TABAS03KIT | |
Equipment | |||
BluPAD Dual LED Blue/White light transilluminator | Bio-Helix | BP001CU | |
Dry Bath Dual Block | ELITE | 4-2-EL-02-220 | Model: EL-02 |
Fluorescence microplate reader | Tecan | 30050303 | Model: Infinite 200 Pro |
Freeze dryer | LABCONCO | 794001030 | FreeZone Triad Benchtop Freeze Dryer |
Microplate 384-well | Greiner | GDE0784076 | F-Bottom, small volume, Hibase, Med. Binding, Black |
Real-time thermal cycler (CFX Connect Real-Time PCR System) | Bio-Rad | 185-5201 | Model: CFX Connect Optics Module |
Oligonucleotide | |||
s-gene forward RPA primer | IDT | GAAATTAATACGACTCAC TATAGGGAGGTTTCAAAC TTTACTTGCTTTACATAGA |
|
s-gene reverse RPA primer | IDT | TCCTAGGTTGAAGA TAACCCACATAATAAG |
|
n-gene forward RPA primer | IDT | GAAATTAATACGACTC ACTATAGGAACTTCTC CTGCTAGAATGGCTG |
|
n-gene reverse RPA primer | IDT | CAGACATTTTGCTCTC AAGCTGGTTCAATC |
|
LwaCas13a-crRNA for the s gene | Synthego | GAUUUAGACUACCCCAAAAAC GAAGGGGACUAAAACGCAGCA CCAGCUGUCCAACCUGAAGAAG |
|
LwaCas13a-crRNA for the n gene | Synthego | GAUUUAGACUACCCCAAAAACG AAGGGGACUAAAACAAAGCAAG AGCAGCAUCACCGCCAUUGC |
|
FAM-polyU-IABkFQ reporter | IDT | 56-FAM/rUrUrUrUrU/3IABkFQ | |
O-hannah_cytb_F RPA primer | IDT | GAAATTAATACGACTCACTA TAGGGTACGGATGAACCATA CAAAACCTTCACGCAATCG |
|
O-hannah_cytb_R RPA primer | IDT | AAGATCCATAGTAGATTC CTCGTGCGATGTGGATA |
|
synT7crRNA13a_ O_hannah | IDT | GCGCATCCATATTCTTCAT CTGCATTTAGTTTTAGTCC CCTTCGTTTTTGGGGTA GTCTAAATCCCCTATAGT GAGTCGTATTAATTTC |