preparation of lyophilized reverse transcriptase-recombinase polymerase amplification (rt-rpa) reactions 

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

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 &#34;collateral&#34; 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 &#176;C), and therefore, has low energy and equipment requirements. Other isothermal amplification reactions require higher temperatures (LAMP, 60-65 &#176;C; strand-displacement amplification (SDA), 60 &#176;C; exponential amplification reaction (EXPAR), 55 &#176;C; and helicase-dependent amplification (HDA), 65 &#176;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 &#176;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&#39; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66703/66703fig01v2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/66703/66703fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

We developing point-of-care test to detecting these material of infectious diseases. This test use isothermal nucleic acid amplification and CRISPR-based detection. With highly osmotic accuracy similar to PCR, they can detect small sequence differences including single nucleotide polymorphism and can be multiples.CRISPR, the osmotic reactions contain multiple components and steps and are difficult to perform at the point of care. As CRISPR-based, the osmotics are highly sensitive, there is a risk of carryover contamination which can lead to false results. Fortunately, this problem can be mitigated if we simplify the procedures of the reactions.We offer an easy to use method to detect our disease pathogens and genetics markers using recombinase isothermal amplification and CRISPR-based reagents. These reagents are simple to store and use. We also offer tips to fix common issues when using these methods and reagents, making it easier for people to use them.The protocol here uses CRISPR reagents, which facilitate the transportation and storage. The freeze-dried reagents can easily be rehydrated at the points of use and retain high amplification and detection efficiencies. We hope to apply CRISPR diagnostic technologies to detect endemic and emerging infectious disease.If this test can be routinely and correctly performed at the point of care, that could inform antiviral and antibiotic treatment options, potentially save lives, reduce drug overuse, and the development of resistance.

Preparation of Lyophilized Reverse Transcriptase-Recombinase Polymerase Amplification (RT-RPA) Reactions 

To begin, incubate the triglycine solution at 60 degrees Celsius. Remove three RPA pellet strip tubes from the freezer. Tap the strip tubes gently against the lab bench to dislodge the white pellets.Transfer all three pellets into a 1.5 milliliter microcentrifuge tube A.Pipette 25 microliters of nuclease-free water to rinse any remaining RPA powder in the strip tubes. Transfer the rinse solution to a new 1.5 milliliter microcentrifuge tube B.Then, add 10 microliters of the 0.57 molar triglycine solution and 10 microliters of primer mix to the master mix tube B.After vortexing and spinning down the tube, place it on ice. Next, add 28.4 microliters of the five times RPA buffer to the master mix tube B.Mix thoroughly by strong tapping or shaking multiple times.Transfer pellets collected in tube A into the master mix tube B and invert the tube or tap the bottom of the tube with your finger several times to mix. Observe whether the solution appears homogeneous and monophasic. Place the tube on ice.Then add 0.71 microliters of reverse transcriptase and 2.84 microliters RNase H to the master mix tube. Gently tap or invert the master mix tube up and down many times. Briefly spin down the tube before placing it on ice.Now, aliquot 8.4 microliters of the master mix solution to 10 pre-cooled 1.5 milliliter tubes and place the tubes on ice. After aliquoting, place the tubes in a metal rack submerged in liquid nitrogen to prepare them for lyophilization. At the freeze dryer, check collector and shelf temperatures.Now, cover open tubes in the metal rack with a sheet of cleaning white paper. Place the metal rack along with the open tubes in the freeze dryer shelf and close the shelf door. Then, select the desired program and press Start.After 30 minutes of the secondary drying step, press stop. Release the pressure by opening the vacuum release valve. After removing the rack from the shelf, cap the tubes immediately.

preparation-lyophilized-reverse-transcriptase-recombinase-polymerase

Research

JoVE Journal

Bioengineering

63 Views. To begin, incubate the triglycine solution at 60 degrees Celsius. Remove three RPA pellet strip tubes from the freezer. Tap the strip tubes gently against the lab bench to dislodge the white pellets.Transfer all three pellets into a 1.5 milliliter microcentrifuge tube A.Pipette 25 microliters of nuclease-free water to rinse any remaining RPA powder in the strip tubes. Transfer the rinse solution to a new 1.5 milliliter microcentrifuge tube B.Then, add 10 microliters of the 0.57 molar t...

Video: Preparation of Lyophilized Reverse Transcriptase-Recombinase Polymerase Amplification (RT-RPA) Reactions 

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.

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 ...

Preparation of Lyophilized CRISPR-Cas13a Premixed Detection Reagents for Nucleic Acid Biomarkers Detection

To begin, prepare the CRISPR-Cas13a master mix reaction using the components shown here. After thoroughly mixing the contents, spin down the master mix tube. Aliquot 17 microliters of the master mix into ten 1.5 milliliter tubes placed on ice.Place the tubes in a rack, submerged in liquid nitrogen, and allow them to remain submerged for five minutes. At the freeze dryer check collector and shelf temperatures, then place the tubes in the freeze dryer. Select the program and press start.After 30 minutes of the secondary drying step, stop the program. To release the pressure open the vacuum release valve and remove the rack from the shelf. Cap the tubes immediately before storing the lyophilized reactions at minus 20 degrees Celsius.

RT-RPA Nucleic Acid Amplification and CRISPR-Cas13 Nucleic Acid Detection

To begin, prewarm a thermal incubator at 42 degrees Celsius. Add one microliter of magnesium acetate and potassium acetate solution mix to the side of the tube containing the lyophilized, pre-mixed RTRPA pellet. Resuspend the pellet in 12.4 microliters of the RNA sample.To initiate the RTRPA reaction, briefly spin down the added solution using a spin down mini centrifuge at room temperature for three seconds. Carefully tap the tube to mix the contents thoroughly and spin down again. Incubate the reaction at 42 degrees Celsius for 60 minutes in a preheated thermal shaking incubator.After the reaction has ended, remove the reaction tubes and place them on ice before proceeding to the CRISPR-Cas detection. For nucleic acid detection, turn on a fluorescence microplate reader. Set and preheat the microplate reader to 37 degrees Celsius and select the program.Then place a 384 well plate on ice. To resuspend the lyophilized CRISPR-Cas pre-mixed detection reaction, add 17 microliters of eight millimolar magnesium chloride solution. Mix by carefully tapping the tube.Briefly spin down for three seconds before placing the tube on ice. Transfer 18 microliters of the resuspended reaction mix to each well of the pre-cooled 384 well plate on ice. Add two microliters of the RPA product prepared to each reaction well.Remove the plate from the ice. Quickly place it into a preheated fluorescence microplate reader and press Start. For lyophilized, pre-mixed Cas13a-based reactions, either 6%trehalose or 2%sucrose preserves the reaction efficiency well after lyophilization.The lyophilized pre-mixed reagents for RTRPA and Cas13-based reactions are stable at 20 degrees Celsius for at least eight months.