Mankind has been fighting infectious diseases for many decades now. A serious threat is posed by those infections that cause outbreaks or epidemics [1]. Because a successful treatment outcome depends in the first place on the accuracy of the diagnosis, a search for novel diagnostic approaches continues. Unlike classical microbiological methods of pathogen identification based on the use of differential culture media, polymerase chain reaction (PCR) ensures rapid detection of microorganisms regardless of the specifics of their life cycle. Evolution of sequencing techniques and free access to public databases containing sequencing data encourage a more active use of PCR [2].

Still, there is a need for novel molecular diagnostic techniques. There are a few obstacles preventing a wider application of PCR, including high equipment costs and a lack of laboratory facilities and qualified personnel. Among the proposed alternatives to PCR are mobile biosensors based on a combination of physical and biological approaches [3–5] and techniques that do not rely on complex equipment [6, 7]. We believe that the most promising technique that provides high specificity and sensitivity for single molecule detection is Specific High-Sensitivity Enzymatic Reporter Unlocking, or SHERLOCK [8, 9].

SHERLOCK combines isothermal amplification of total nucleic acids and Cas13a activity, allowing detection of both DNA and RNA molecules. Isothermal amplification ensures accumulation of target molecules, while Cas13a acts as a sensor capable of accurate target recognition, including single nucleotide polymorphisms [9].

Cas13a nuclease activity is initiated when CRIPSR guide RNA (crRNA) binds to Cas13a entailing significant conformational changes in the protein structure aimed to form a channel for the binding of a target RNA [10]. When Cas13a “meets” target RNA, a guide crRNA/target RNA duplex is formed in a positively charged NUC lobe channel. The target RNA serves as an activator: duplex formation catalyzes the movement of catalytic domains towards each other, followed by the formation of an RNA cleavage site. The use of RNA probes enables a visual representation of fluorescence signal accumulation as Cas13 exerts its activity.

The aim of this work was to obtain a functionally active Cas13a protein and study its behavior under different conditions, including varying concentrations of a target molecule represented by an RNA fragment of the influenza B virus.

METHODS

Synthesis of the recombinant protein LwCas13a

To obtain the recombinant protein Cas13a of Leptotrichia wadei (LwCas13a), a codon-optimized gene synthesized de novo by Evrogen, Russia, was incorporated into the gene expression vector pET42b(+) under the control of lacТ7 promotor. Gene expression was induced in the cells of Escherichia coli BL21(DE3) pLysS, driven by isopropyl β-D-1-thiogalactopyranoside (IPTG). The resulting Cas13a protein was tagged with octo-histidine on its C-terminus. The frozen bacterial cells were resuspended in the lysing buffer (20 mM Tris HCl, pH of 8.8, 500 mM NaCl, 5 mM β-mercaptoethanol) and lysed by exposure to cyclic pulsed ultrasound. The lysate was centrifuged for clarification at 15,000 g for 20 min; the supernatant was then used for the affinity chromatography on automated medium-pressure system NGC Discover^тм 10 (Bio-Rad, USA) with the 20 ml HisPrep FF 16/10 column (GE, Germany) pre-charged with Ni²⁺ ions. To remove non-specifically bound impurities, Triton X-100 was added to the buffer solutions at a final concentration of 0.1%. The protein was eluted using a linear imidazole gradient (the final concentration was 0.5M). After chromatography, Cas13a-containing fractions were combined and dialyzed against a storage buffer (20 mM Tris HCl, pH of 8.0, 200 mM NaCl, 0.1 mM EDTA). Protein concentrations were measured spectrophotometrically at 280 nm wavelength using Implen NanoPhotometer (IMPLEN, Germany). Concentrations were calculated accounting for the extinction coefficient [11, 12].

Acquisition of target and guide RNAs

Guide and target RNAs were obtained through PCR followed by the transcription of PCR products using the qPCRmix-HS SYBR kit (Evrogen, Russia) according to the manufacturer’s protocol. Guide RNA was obtained using artificially synthesized oligonucleotide primers with self-complementary regions. To get a target RNA molecule, we amplified a plasmid fragment carrying a sequence of the influenza B virus and a sequence of the MS2 phage. In vitro transcription of the amplicons was aided by the MEGAscript® T7 Kit (Thermo Fisher Scientific, USA).

Testing LwCas13a for non-specific nuclease activities

To test LwCas13a for non-specific nuclease activities, we measured fluorescence. Briefly, the fluorescence signal is emitted when the reporter RNA molecule RNAseAlert v2 Substrate (Thermo Fisher Scientific, USA) is cleaved. The reporter RNA molecule is an oligonucleotide beacon with a fluorescent dye sitting on its 5′-end and a quencher on its 3′-end. When the molecule is cleaved, the dye is separated from the quencher emitting light in the green spectrum at 520 nm wavelength. The final LwCas13a concentration of 450 nM was incubated at 37 °C for 2 hours in the reaction mix containing a nuclease buffer and the reporter RNA (40 mM Tris HCl, pH of 7.3, 60 mM NaCl, 6 mM MgCl₂, 125 nM RNAseAlert v2 Substrate); fluorescence was measured in real time on the QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA). RNAse A (Thermo fisher scientific, USA) was was used for positive control; pure reporter RNA, for negative.

Testing LwCas13a endonuclease activity

The reaction mix for testing LwCas13a endonuclease activity consisted of a nuclease buffer (40 mM Tris HCl, pH of 7.3, 60 mM NaCl, 6 mM MgCl₂), 450 mM LwCas13a, 22.5 nM crRNA, 125 мМ RNAseAlert v2 Substrate, 2 µl RiboLock RNAse Inhibitor, 100 ng RNA of a tobacco mosaic virus (for the background), and different concentrations of target RNA. Fluorescence was measured in real time for 2 hours at 37 °С using the QuantStudio 5 Real-Time PCR System (Applied Biosystems, USA).

RESULTS

Purification of the recombinant LwCas13a

The recombinant protein LwCas13a was obtained using affinity chromatography. Chromatography products were analyzed by denaturing electrophoresis, which revealed that induced E. coli cells had produced a water-soluble protein with a molecular weight comparable with the predicted LwCas13a weight (139.8 kDa) (fig. 1).

Optimization of reporter RNA (RNAseAlert) concentrations in the reaction mix

To achieve optimal fluorescence intensity, we conducted a series of model tests using RNAse A (fig. 2) and selected the substrate concentration of 125 nM for further experiments. At this particular value the maximal dynamic range of 100, 000 arbitrary fluorescence units was provided.

Testing LwCas13a for non-specific RNAse activities

The obtained LwCas13a protein was tested for non-specific RNAse activities by 2-h incubation with the fluorescent RNAseAlert v2 Substrate in the absence of crRNA and target RNA. The fluorescence signal was not changed during incubation. For positive control, we incubated the fluorescent substrate with RNAse A; for negative control, the reporter RNA was incubated without any additives (fig. 3). We found that our method yielded the LwCas13a protein that did not exhibit any non-specific RNAse activity, which allowed us to proceed to the study of characteristics of its pre-programmed RNAse activity.

Testing method sensitivity using the influenza B virus

Our LwCas13a-based detection method was tested for sensitivity in a series of model tests, for which we employed a fragment of influenza B viral RNA sequence, which served as a target. The lower sensitivity threshold observed was 10⁷ molecules of viral RNA (fig. 4 and fig. 5).

DISCUSSION

LwCas13a-based pathogen detection opens up new diagnostic horizons. In 2017 Cas13 was adapted for the use in a platform called SHERLOCK [7, 9]. The latter combines Cas13a-based detection of RNA targets pre-amplified by recombinase polymerase amplification (RPA) and Т7-transcription. All reactions take place in the same reaction mix. Using this approach, the researchers were able to design a diagnostic platform for Zika detection. Its attomolar sensitivity and specificity proved to be no less inferior to those of quantitative PCR (qRT-PCR) and droplet digital PCR. The researchers studied Cas13a orthologs to obtain a stable and reliable fluorescent signal emitted when Cas13a started to exert its RNAse activity. They found that Cas13a of Leptotrichia wadei was capable of detecting up to 50 pM of target RNA [7]. So, this enzyme served as a basis for the SHERLOCK platform. Although the sensitivity demonstrated by Cas13-based detection was high, the researchers decided to investigate a possibility of combining it with different types of isothermal amplification and established that RPA combined with transcription and the effect of Cas13a RNAse activity could improve the sensitivity of the method even more. SHERLOCK is able to discriminate between target RNAs that differ in only one nucleotide and are present in the solution at very low concentrations; the platform can also be used as a portable tool [7, 9].

In our research work we have synthesized a codonoptimized variant of LwCas13a. Unlike the previously described protein [7, 9], our protein had a different sequence changed for more effective expression in E. Coli cells. The protein yield was up to 10 mg from 1L of culture (fig. 1). We have also elaborated a method for Cas13a purification and tested it to discover that programmed RNAse does not exhibit any non-specific RNAse activity. We ran a few in vitro tests to evaluate the specificity of Cas13 RNase activity against the fragments of viral RNA. Without target pre-amplification by previously described RPA and T7-transcription [7, 9], the sensitivity of our technique was 10⁷ molecules per reaction.

CONCLUSIONS

In the course of this work we synthesized a codon-optimized variant of Leptotrichia wadei Cas13a that exhibits specific RNAse activity in E. coli cells. The obtained protein is deprived of non-specific nuclease activities and can specifically detect the target, which in our case was a fragment of influenza RNA. Further research will be aimed at perfecting the technique, improving its sensitivity, studying programmed RNAse specificity, and increasing multiplexity. Besides, we will attempt to create a Cas13a-based diagnostic system for portable use. Field diagnostic tools can be of great assistance in monitoring the agents of infection in their natural reservoirs, preventing the invasion of pathogens into the human population [13–15].