CRISPR-Powered Microfluidics Applications

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1		Patricia Khashayar	+ 3600 word(s)	3600	2021-12-29 09:40:43	\|
2	format correct	Conner Chen	Meta information modification	3600	2022-01-10 01:52:48	\|

This entry is adapted from the peer-reviewed paper 10.3390/chemosensors10010003

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a simple yet powerful tool for highly specific and rapid modification of DNA in a genome, which is the complete set of genetic instructions in an organism. One of the requirements for successful modification of the eukaryotic genome using the CRISPR/Cas9 system is the presence of the guide RNA (sgRNA or crRNA/duplex crRNA/tracrRNA) Cas9 protein complex and the introduction of mRNA or DNA. In therapeutic approaches, the genome of specific cells can be re-transplanted into the patient in vitro and then the host genome modified to treat any possible deficiency in genes. Nowadays, the use of microfluidic channels and chips is one of the best approaches to deliver materials and cells as it prevents many problems by accurately editing the cell and creating an opportunity for successful editing and screening of their genome. Such chips provide a suitable substrate for cell manipulation, drug screening, and exosome characterization. Furthermore, they are useful for pathogen and cancer detection because of their high throughput, low cost, flexibility, and controlled fluid or gas flow.

microfluidics lab-on-chip CRISPR biosensor medical detection

1. Microfluidic CRISPR-Based Biosensors for Virus

1.1. SARS-CoV-2

The COVID-19 pandemic pointed out the importance of rapid, accurate, and on-demand detection of viruses such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is still the gold standard test for the detection of this positive single-stranded RNA (ssRNA) virus responsible for more than 4.8 million deaths worldwide ^[1]. This is while rapid detection tests based on the clustered regularly interspaced palindromic repeats of (CRISPR)/Cas proteins in combination with microfluidics are shown to be new promising approaches for pathogen detection ^[2].

In this regard, Silva et al. developed a cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay (Figure 1A) ^[3]. After RNA extraction from nasal swabs, the prepared sample was combined with reverse transcriptase (RT) for about 10 min to produce RNA/DNA hybrids. Afterward, the catalase-ssDNA probe bonded to the magnetic beads, and CRISPR/Cas12 protein was added to the reaction chamber for 1 h. Following the specific binding of CRISPR/Cas12 to the SARS-CoV-2 genome, the trans cleavage ability of Cas12a caused the cleavage of ssDNA and production of free catalase. After separating the catalase-ssDNA probes using magnetic beads, 6% hydrogen peroxidase solution was added to generate bubbles, which could be recorded by a smartphone camera. The optical signal generated by these bubbles appeared in 1 min and was simply and quantitatively measured by the smartphone without any need for external devices. CASCADE was capable of detecting down to 50 RNA copies per µL without any amplification; this capacity was reduced to five copies per reaction when an amplification step was added. It was concluded that CASCADE is a smartphone-based amplification-free detection platform with the minimum hardware needs. Removing the extraction step and providing an artificial intelligence (AI)-based image databank can help provide faster and more sensitive detection results.

Figure 1. (A) CASCADE assay based on the trans cleavage activity of Cas12, in which the catalase enzyme produces gas bubbles for visual detection ^[3]. (B) deCOViD assay in which fluorophore-quencher probes break after the activation of Cas12. Cas12 trans cleavage activity and the microfluidics provided the digitized fluorescent signals ^[4].

Another smartphone-based pathogen detection resource multiplier was developed using adversarial networks (SPyDERMAN) for intact and viral nucleic acid detection ^[5]. The CRISPR/dCas9 protein used in this platform has catalytical inactivated cas proteins that selectively bind to the target sequence without any endonuclease activities. This platform, using a dataset library of adversarial neural network algorithms, could specifically detect five different viruses. These algorithms helped not only to standardize the shape and size of bubbles but also to reduce the differences in various phone cameras. An example of reaction fundamentals for SARS-CoV-2 will be discussed later in the paper. The microfluidic platform was made of poly (methyl methacrylate) sheets, which is a great idea for automated applications with lengthy extraction and amplification processes requiring smaller amounts of reagents. After RNA extraction and cDNA amplification, the mixture was added to a CRISPR/dCas9 solution. After proper incubation, platinum nanoparticles (PtNPs) bonded to monoclonal antibodies against Cas9 and a 6% hydrogen peroxidase solution was added. Later, the gas bubbles produced by the catalase-like activity of PtNPs were photographed.

A new CRISPR-based sensor known as digitization-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOViD) was developed by Park et al. (Figure 1B) ^[4]. deCOViD is based on reverse transcription and recombinase polymerase amplification (RT-RPA), which uses fluorophore probes like Alexa647. After the production of DNA amplicons, CRISPR/Cas12a specifically cleaved the target DNA so that the fluorescent signals could be observed. Integrating this into a digitized microfluidic chip, besides minimizing the reagents and materials, increased the reaction speed thanks to the locally high concentration of nucleic acids in the small microfluidic wells. Using RT-RPA instead of traditional RT-PCR and transforming the platform into a microfluidic platform also reduced the detection time by up to 15 min. The system was also capable of reading the heat-inactivated virus without extracting RNA in 30 min. Since RNA extraction and purification were no longer needed, the detection time became shorter and LOD improved. CRISPR/Cas12a with a proper crRNA used in this research was responsible for the high specificity of the sensor. deCOViD has the lowest LOD in comparison with similar CRISPR-based platforms. The LOD was reported to be one genome equivalent (GE) per µL for SARS-CoV-2 genome detection and 20 GE per µL for the heat-inactivated SARS-CoV-2 samples.

A novel SARS-CoV-2 POC, introduced by Chen et al., composed of miniaturized magnetic arms, fluorescent detector, and heating module (Figure 2A) ^[6]. The sensor known as POC-CRISPR worked in three main steps. Firstly, a nasopharyngeal swab was inserted into an elution tube containing detergent and magnetic beads buffers. The detergent caused virus decomposition and then the negatively-charged RNAs bonded to the positively-charged magnetic beads. Next the solution was transferred to the working cartridge and was placed in the droplet magneto-fluidic (DM) device, where the extraction, purification, and concentration processes happened all together. In this step, the magnetic beads separated the RNAs from other cell lysates and concentrated them by moving them to a new area in the microfluidic device. Following these processes, the purified RNA was produced using the RT-RPA and DNA amplicons. In this step, fluorescent signals were generated when fluorophore probes were released through DNA cleavage by the CRISPR/Cas12a. The signals were then detected by the DM device, and signals were wirelessly sent to a cellphone. The microprocessors embedded in the DM device accelerated this step. The POC-CRISPR was reported to have an acceptable LOD (1 GE per µL), require a low (100 µL) starting solution, and have a short duration of less than 30 min. In this system, the magnetic beads acted as the fundamentals for RNA purification and concentration along with shortening the purification time; they also enhanced the sensitivity thanks to their concentrating ability. Therefore, their applicability in high-tech sensors to increase the performance of CRISPR-based assays, especially in microfluidic devices, was shown.

Figure 2. (A) POC-CRISPR assay with droplet microfluidics, in which RNAs are separated and concentrated using electrostatistical attachment to magnetic beads and fluorescent signals again using Cas12 trans cleavage ^[6]. (B) An electrokinetic microfluidic chip that creates isotachophoresis mobility in the microfluidic device for RNA separation and sensitivity enhancement ^[7].

Electrokinetic microfluidic sensor coupled with CRISPR/Cas12 and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is another example (Figure 2B) ^[7]. This sensor was modified for SARS-CoV-2 detection using isotachophoresis (ITP) technique and an electrokinetic microfluidic chip. First, ITP-based ion mobility was generated in the microchannel embedded in the chip. The coexistence of a high-mobility leading (LE) and a low-mobility trailing (TE) buffer in a microfluidic channel resulted in different ion mobility in the sample following the application of an electric field. This phenomenon caused negatively charged nucleic acids to be fully separated from other parts of a cell lysate. After the nucleic acid separation and purification steps in a nasopharyngeal sample, the SARS-CoV-2 RNA genome was transformed into cDNA using the RT-LAMP system in the chip. After the amplification process, CRISPR/Cas12 specifically recognized the target sequence. Then ssDNA, which was attached to a fluorophore and a quencher, was cleaved by the trans cleavage ability of the CRISPR/Cas12 protein. By separating the fluorophore and the quencher pair, the fluorescent signals representing the desired genome, this process happened in 30–40 min with an LOD of 10 copies per µL. Automated reagent mixing, need for a low amount of reagents, and accurate detection are the main advantages of this sensor.

Puig et al. developed a fluorescent sensor called miSHERLOCK (minimally instrumented SHERLOCK). It worked based on the trans cleavage ability of the CRISPR/Cas13 ^[8]. This specific enzymatic test was implemented in a miniatured device that extracted and concentrated the viral RNA, needed for SARS-CoV-2 detection, in the saliva samples. The device consisted of two parts. In the first part, the saliva sample was inserted, and then the RNA molecules were extracted and concentrated using polyethersulfone (PES) membrane. After this step, the concentrated RNA met the CRISPR/Cas13 enzyme and other related cocktail factors in the second part of the two-chambered holder containing the fluorescent laser. Around 1 h later, the device was connected to a mobile as the fluorescent readout. This platform could detect three different variants of SARS-CoV-2 successfully with an average LOD of 1200 copies per mL for the universal variant. Albeit the sensitive and smart idea behind the miSHERLOCK, the need for 4 mL of saliva sample and the 55-min reaction time were among its shortcomings.

1.2. Ebola Virus

Ebola is a deadly virus from the Filoviridae family with a negative ssRNA genome. It was responsible for a deadly outbreak of a hemorrhagic fever syndrome between the years 2013 and 2020 ^[9]. While sequencing and PCR methods are the main detection methods of Ebola virus, POC devices with a need for minimum reagents and inexpensive materials seem to be required for possible future endemics.

A POC sensor for Ebola detection based on the CRISPR/Cas13a trans cleavage ability was developed by Qin et al. ^[10]. In this attempt, a microfluidic chip with 24 parallel assays for the Ebola RNA and CRISPR-crRNA combinations was fabricated. During the free-amplification process, CRISPR/Cas13a-crRNA targeted the Ebola genome and after Cas13 activation, due to the trans cleavage ability of the Cas protein, the quenched RNA probes were cleaved, resulting in fluorescent signals. The reported LOD was 5.45 × 10⁷ copies per mL in 5 min. The inexpensive POC sensor needed low sample volume. The short detection time, little background signals, and the free-amplification process were among its advantages. The high cross-reactivity reported in this platform could be due to the off-targets of CRISPR/Cas13a, and could be reduced by developing recombinant Cas’s proteins with fewer off-targets.

Smart nucleic acid response materials [PEG hydrogel with ssDNA linkers (PEG-ssDNA) and polyacrylamide hydrogel with DNA linkers (PA-DNA)] could also be fabricated for this purpose. PEG-ssDNA hydrogels absorb fluorophores and enzymes, while PA-DNA hydrogels entrap gold nanoparticles (AuNPs) and cells. When CRISPR/Cas12a proteins in the hydrogel structure meet the target sequence, their trans cleavage activity breaks the DNA linkers, changing the morphology of the hydrogels. It also results in the release of the fluorophores bonded to the linkers or entrapped AuNPs, producing color signals. Based on these fundamentals, a paper-based microfluidic system (µPAD) was fabricated for Ebola detection ^[11]. The target genome was amplified by RT-RPA and inserted into the µPAD with embedded hydrogels. Based on the lateral flow (LF) mobility of the reaction molecules, different signals were obtained. The color- (fluorescent signals for the fluorophores, and visual ones for the AuNPs), electric- (by combining the µPAD system with an electrode-based chip for conductivity measurement), and visual microscopy-(morphological changes of hydrogels) based signals are various output signals leveraged from the described microfluidic system. Although the sensor could detect the Ebola genome at concentrations as low as 11 aM, the hydrogel alteration process was time-consuming and thus the response signals only appeared after a few hours, which is not suitable for rapid detection tests. Furthermore, due to the sensitivity of CRISPR/Cas proteins to temperature, this material may not work properly for long hours at room temperature.

1.3. Human Immunodeficiency (HIV)

HIV is a positive ssRNA virus that belongs to the Retrovirus family. The virus causes human acute immunodeficiency syndrome (AIDS) and has been responsible for a global pandemic with almost 30 million deaths during the past four decades. Although lifelong antiretroviral therapy (ART) is still its main treatment, there is still no efficient drug or vaccine on the market. As a result, fast and reliable detection methods remain crucial for such widespread syndrome to reduce its complications ^[12].

A sensor for C-C chemokine receptor type 5 (CCR5) gene detection related to the human immunodeficiency virus (HIV) was developed as a model by Lee et al. (Figure 3A) ^[13]. This gene expresses one of the most important entry receptors for HIV. The sensor contained a microfluidic platform with a microchannel creating ion concentration polarization (ICP). The detection process in the sensor was based on different mobility behaviors monitored by fluorophores. Following the ICP generation in the microchannels and based on different hydrodynamic behavior of negatively charged moieties that are targeted DNA in this case, two different mobility behaviors were reported. In the presence of free DNA (non-target sequence) in the microchannel, the propagation mobility behavior was reported. This is while the stacking mobility was observed in the presence of DNA-CRISPR/dCas9 (target sequence), which was later coupled with fluorophores (FAM and cy3). The sensor was able to detect 3 pM DNA in 100 min by a 18.4 pM/min concentration rate. It, however, has not been tested on real physiological samples, as the development of proper ICP in such complex samples may be challenging or affected by different factors. Moreover, unbalanced DNA and CRISPR/dCas9 molar ratios (for example, in the presence of very low or very high amounts of target DNA in the sample) could lead to false-negative and overload responses, respectively. Additionally, the preconcentration process reported along with the stacking behavior overcame the need for amplification in this sensor.

Figure 3. (A) The microfluidic platform with a microchannel creating ion concentration polarization (ICP) shows two different mobility behaviors of bound and unbound DNA-CRISPR complexes. Green and orange colors represent the stacking and propagation mobility behaviors, respectively ^[13]. (B) The DAMR platform represents the dynamic diffusion of cleaved nucleic acid products after Cas12 trans cleavage activity through the multiphase sucrose solutions with different concentrations ^[14].

1.4. Human Papillomavirus (HPV)

HPV is a circular double-stranded DNA widespread virus with nearly 200 subtypes. HPV infects basal epithelial cells, leading to certain cancers such as cervical cancer. More than 90% of cervical cancers are believed to be HPV related. HPV16 and HPV18 are the most common subtypes, mainly detected through sequencing or PCR methods ^[15]. Despite the approval of a vaccine against HPV, early detection using rapid and non-invasive methods is still needed and yet challenging.

In an attempt by Yin et al., a POC device consisted of a three-chambered-microfluidic chip and a dynamic aqueous multiphase reaction (DAMR) system conjugated with RPA (for amplification), and CRISPR/Cas12a (for selective detection) was developed (Figure 3B) ^[14]. The dynamic aqueous multiphase was generated using different sucrose concentrations: DNA sample in the 40% sucrose concentration in the bottom phase, RPA reagent in 10% sucrose in the middle phase, and CRISPR/Cas12a with fluorophore-quencher DNA probes in the top phase. In this regard, DNA was dynamically diffused from the bottom phase (with higher sucrose concentrations) to the top one (with lower sucrose concentrations). It was amplified in the middle phase with RPA before being diffused to the third phase, where the fluorescent signals appeared thanks to the trans cleavage properties of CRISPR/Cas12a. In this phase, the fluorophores were released from the fluorophore-quencher probes following the selective DNA binding to the crRNA. This sensor detected HPV16 and HPV18 with 10 and 100 copies per mL, respectively, in 1 h. The use of multiphase dynamic mobility reduced the reaction time by about 100 times compared with the single-phase reaction methods. This novel multiplex sensor was easily fabricated. Pretreatment steps like extraction and purification, however, are still needed for real samples.

2. Microfluidic CRISPR-Based Biosensors for Bacteria Detection

CRISPR-based sensing devices are also investigated in bacteria detecting sensors. In an attempt, Chen et al. developed a sensor for Pseudomonas aeruginosa genome detection (Figure 4A) ^[16]. This bacterium, which exists in the endothelial layers, is responsible for several illnesses in humans. The fundamental detection principle of Cas12a-assisted microfluidic equipment for nucleic acid analysis (CASMEAN) is the generation of fluorescent signals after the trans cleavage of fluorophore probes and the cis cleavage of the target genome by CRISPR/Cas12a. All the required reagents were freeze-dried in the microfluidic disc under nitrogen gas for long storage possibilities. The centrifugal microfluidic disc used in this system consisted of a polymethyl methacrylate (PMMA) layer with 32 reaction chambers and was surrounded by two sealing membranes. When a sample was loaded onto the disc, the reactions happened automatically. Firstly, the DNA genome was amplified by the recombinase-aided amplification (RAA) process and then CRISPR/Cas12a trans cleavage caused the production of fluorescent signal in the same chamber. The logic behind this phenomenon was the amplicons being rapidly cleaved by the CRISPR/Cas12a before enough trans cleavage happened. In this way, the sensitivity was low as the amplification and detection parts were not separated. It should be noted that the RAA protected the enzymatic structure of the CRISPR/Cas12a protein more than PCR and LAMP methods due to its lower and more stable thermal cycles. Furthermore, the concentration of CRISPR/Cas12a in the chamber was optimized to reduce the risk of amplicons deterioration and improve the sensor’s sensitivity. The sensor was reported to have a low LOD (10 aM or 103 PFU/mL) with a 90 min turnaround time. Besides its low LOD, being fully automated and requiring low amounts of sample and reagents were other advantages for this sensor.

Figure 4. (A) The CASMEAN assay with a centrifugal microfluidic disc containing the freeze-dried CRISPR/Cas12 for long-term applications and fluorescent signal readouts ^[16]. (B) The CARMEN multiplex detection assay for 169 viruses is based on the colored map analysis created by four commercial fluorophores and Cas13 trans cleavage in a microarray chip ^[17].

3. Microfluidic CRISPR-Based Biosensors for Multiple Species

Combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN) combined with the CRISPR/Cas13 was developed by Ackerman et al. for the detection of a wide range of viruses (Figure 4B) ^[17]. In this attempt, the desired viral genomes were amplified using suitable primers with PCR or RPA methods. Then, the amplified samples were inserted into the same pool and combined. The prepared samples and droplets containing CRISPR/Cas13 with reporter probes were thereafter injected into the microarray microfluidic chip. Afterwards, the two droplets (amplified samples and CRISPR/Cas13 with reporter probes) were combined in all possible paired formats. The microfluidic chip was made of polydimethylsiloxane (PDMS) using soft lithography and provided an array for simultaneous detection of thousands of viral genomes. Reporter probes were made of four available commercial fluorophores, which could generate about 1050 different colors. The high number of colors led to different colored maps in the microarray chip under fluorescent microscopy. Computational biology and color algorithm interpretation techniques were key in this detection platform, which could perform massive detection (around 4500 tests) in a single chip. The low cross-reactivity with high reliability observed in the chip may be due to the highly selective nature of the used CRISPR systems and the monitoring system, which reduced the chance of error in each array. The smallest nucleic acid used in the sensor was reported to be 10⁴ copies per µL. Besides being capable of detecting 169 viral genomes, different Influenza A subtypes and HIV mutants were also distinguished with this tool. This shows the accurate detection ability of the sensor even for small changes and variants. This platform was also used for the detection of newborn pathogens such as SARS-CoV-2, enabling the possibility of rapid adaptations to new targets.

Zika (ZIKV) and Dengue (DENV) viruses have been considered as global health emergencies by the world health organization (WHO) since 2016. They both have a positive ssRNA genome, belonging to the Flavivirus genus, and are responsible for mild to severe symptoms. Zika causes Guillain–Barré syndrome and is associated with microcephaly, while Dengue virus causes mild to hemorrhagic fever ^[18]. New insights in to PoC detection of these two viruses using the microfluidic-based CRISPR will be discussed here.

A LF nucleic acid detection test was developed by Gootenberg et al. ^[19]. SHERLOCK (specific high sensitivity enzymatic reporter unlocking) was designed to detect the ssRNA genome of Zika and Dengue viruses through trans cleavage activity of CRISPR/Cas13. After RNA amplification using RPA, the sample was loaded on a LF assay, where these two viruses were detected at 2 aM under 90 min. The reported LF consisted of sample, control, test, and absorption pads. When the sample was loaded on the assay paper, the trans activity of CRISPR broke the nucleotide link between the FAM and biotin in the presence of the targeted RNA. Then they were exposed to the anti-FAM antibody conjugated with AuNPs on the test pad. In the absence of the target RNA, the AuNP-conjugated antibody bound to FAM and then to the control band via the biotin–streptavidin connection. In this regard, few FAM remained available for the second band to be seen. In the presence of the target RNA, the nucleotide link between the biotin and FAM broke, and the FAM reporters bonded to the second band along with the AuNP-conjugated antibody, resulting in a red-colored band. Furthermore, the self-fluorescence properties of the FAM reporter were also traceable.

References

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