Posted by on February 12, 2021 7:10 am
Categories: CRISPR-Google

CRISPR/Cas technology as a promising weapon to combat viral infections – Wiley

Nucleic acid detection has been extensively used for molecular diagnosis of viral infections, mainly by quantitative polymerase chain reaction (qPCR). This is a highly specific and sensitive method, but requires sample preparation, high‐cost equipment and materials, and specialized technicians, being unfeasible as point of care (PoC) diagnosis or in developing countries. Pandemics like the current SARS‐CoV‐2 outbreak highlight the need for fast, sensitive, and specific PoC virus detection methods. In this regard, the CRISPR/Cas system has great potential as an alternative sensing tool, due to its high sensitivity and specificity (Figure 4). Cas9, Cas12, and Cas13 have been used for viral nucleic acids sensing. Cas9 detection read‐out is based on target recognition and cleavage, while Cas12 and Cas13‐based sensors use their trans‐cleavage activity that is activated upon target recognition. The three CRISPR nucleases are especially suited for viral genotypes discrimination, due to the high specificity of the crRNA target can be abrogated just by changing a small number of nucleotides. However, some limitations hamper its general use for genotype discrimination. Cas9 and Cas12 need the presence of PAM sequences in the target for further recognition (Figure 1).[3, 5] The genotype differences must then be located close to PAM sequences. Some Cas13 orthologs, like Leptotrichia wadei (Lwa) Cas13a, completely lack this kind of sequence requirements.[8] However, Cas13 recognition is highly dependent on targets’ secondary structure. Target RNA secondary structure must be carefully studied to design successful crRNAs and several guides must be tested for optimal detection. Despite these limitations, many relevant CRISPR‐based sensors have been developed to detect viral nucleic acids (Table 3).

Cas9‐based sensors

Cas9’s target recognition and cleavage were combined with isothermal amplification to develop two detection methods: CRISPR‐Cas9‐triggered nicking endonuclease‐mediated strand displacement amplification (CRISDA)[96] and NASBA‐CRISPR.[95]

The CRISDA method uses a pair of engineered Cas9 nickases that produce single‐stranded breaks (nicks) on the target dsDNA. The cleavage triggers the strand displacement isothermal amplification of the target. This isothermal DNA in vitro amplification method uses a DNA polymerase together with restriction enzymes and different probes. The resulting amplicon is detected by fluorescence measurement, using a peptide nucleic acid (PNA) labeled with biotin and Cy5, which is complementary to the middle region of the amplicon. The complex is isolated by streptavidin‐coated magnetic beads and the fluorescence is recorded. This method presents attomolar (aM) sensitivity and single‐nucleotide specificity.[96]

NASBA‐CRISPR is a low‐cost detection system for ZIKV and DENV, two deleterious ssRNA viruses present in developing countries. The system is assembled in a paper‐based platform and combines isothermal RNA reverse amplification, the ability of Cas9 to cleave specific target DNA and toehold switch sensors.[95] Toehold switch sensors are programmable synthetic RNAs that repress gene translation. The binding of a trans‐acting trigger RNA, complementary to the toehold, reverts this repression. When they are coupled with the translation of the LacZ enzyme, the RNA trigger interaction can be detected by the change in color of a yellow substrate. In the NASBA‐CRISPR, the isothermal amplification of the viral RNA produces a dsDNA sequence comprising a trigger sequence, a T7 promoter, and a PAM sequence. The toehold switch sensors are responsible for virus detection, while Cas9’s specificity allows discriminating between different virus strains because the specific DNA cleavage generates a truncated reverse transcribed RNA product that lacks the trigger RNA and is unable to activate the toehold switch sensor, lacking then the substrate color change.[95]

Both methods are sensitive, specific, and suitable for PoC viral nucleic acids detection. The use of isothermal amplification instead of PCR increases their portability since a thermocycler is no longer required. Furthermore, fluorescence and color changes can be detected by benchtop fluorometers or colorimeters. However, these methods are highly dependent on the presence of PAMs in the target and on complicated designs of PNAs and toehold switches. In consequence, Cas9 detectors have been overtaken by Cas13 and Cas12‐based sensors.

Cas12 and Cas13‐based sensors

The success of Cas12 and Cas13‐based sensors lays on their multiple‐turnover trans‐cleavage activity. It is specifically activated by target recognition and allows signal amplification by adding reporter oligonucleotides that are substrates for the trans‐activity. The recognition of one target molecule activates then the cleavage of many reporter oligonucleotides. Both Cas12 and Cas13‐based sensors are experiencing impressive and parallel development since 2017. Additionally, the existence of a dsDNA detector (Cas12) and an ssRNA detector (Cas13) expands sensing possibilities. Cas12‐based sensors are more economically viable since reporter ssDNA oligonucleotides are cheaper than ssRNA. Furthermore, isothermally amplified samples do not need to be transcribed to RNA. However, Cas12 target recognition depends on the presence of PAM sequences in the target. The use of LwaCas13a, which has not sequence restriction recognition, allows the potential detection of any ssRNA target. Nevertheless, the secondary structure of the target RNA must be studied to design suitable crRNA guides.

The first developed Cas13 and Cas12 detectors, named Specific High‐Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK)[103] and DNA endonuclease‐targeted CRISPR trans reporter (DETECTR)[6] are based on the specific cleavage of previously amplified viral nucleic acid. This recognition triggers the trans‐cleavage of quenched ssRNA or ssDNA fluorescent reporters, respectively. The fluorophore is then released, resulting in an increase in fluorescence (Figure 4). Both sensors have been used for the detection of several viruses with aM sensitivity[103, 108111] (Table 3), including SARS‐CoV‐2.[108, 111] In fact, in May 2020 the FDA authorized the use of SHERLOCK for COVID‐19 detection, and it has recently been clinically validated.[112] SHERLOCK is a DNA and RNA detection platform based on the trans‐cleavage activity of LwaCas13a. It requires previous isothermal recombinase polymerase amplification (RPA) or RT‐RPA of the extracted nucleic acids and T7 RNA transcription to convert amplified DNA to RNA. Then, LwaCas13a RNP recognizes the amplified RNA target (Figure 4A). Similarly, DETECTR involves prior RPA isothermal amplification but does not need T7 RNA transcription, as the dsDNA is directly recognized by Lachnospiraceae bacterium (Lb) Cas12a RNP (Figure 4C). In both cases, the fluorescence readout of the signal hampers their use as PoC detectors, especially for developing countries where benchtop fluorometers may not be affordable. However, their strength lies in multiplexed nucleic acid detection, combining RNPs with different specificities and fluorescent probes. SHERLOCKv2[104] took advantage of the large diversity of dinucleotide cleavage motif preferences among Cas13 orthologs to perform four‐channel single‐reaction multiplexing detection. They combined PsmCas13b, CcaCas13b, LwaCas13a, and AsCas12a RNPs that interact with different targets, with reporter oligonucleotides labeled with four different fluorophores, each of them specific to one type of nuclease (Figure 4A). Recently, Sabeti’s group developed a new SHERLOCK‐based multiplexed detection platform called Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acid (CARMEN). CARMEN uses arrays of nanoliter droplets containing the amplified sample, LwaCas13a, and detection reagents. Fluorescence microscopy allows to detect aM concentrations of more than 4500 nucleic acids from different strains of ZIKV, DENV, HPV, HIV, influenza virus, or SARS‐CoV‐2.[106]

SHERLOCK and DETECTR have also been coupled to lateral‐flow readout systems, to develop fully portable PoC detectors.[104, 97] In SHERLOCKv2, the ssRNA reporter is labeled with FAM and biotin, while the paper strip contains gold nanoparticles (AuNPs) coated with anti‐FAM antibodies and two retention lines: a first streptavidin line for biotin binding and a second protein A line for antibody recognition. Reporter cleavage results in the retention of AuNPs on the protein A line instead of in the streptavidin line (Figure 4A). The Cas‐Gold system relies on the same readout, but using LbCas12a and ssDNA oligonucleotides.[97] Interestingly, Barnes et al. have recently developed a mobile phone application that enables the quantification of the viral load from the paper strip based on the band intensity.[110]

Although fluorescence and colorimetric lateral flow are the most used readout methods for Cas12 and Cas13 target recognition, other approaches for signal detection have been explored. English et al. combined microfluidics and CRISPR to detected RPA‐amplified Ebola virus RNA with aM sensitivity. They used Cas12a, DNA hydrogels, and a microfluidic paper‐based analytical device. The DNA hydrogel, formed by acrylamide polymers crosslinked by ssDNA linkers, is located in the microfluidic path, obstructing the flow. The presence of Cas12a RNP and the target DNA triggers ssDNA linkers’ cleavage, opening the flow path. The use of colored fluids allows for naked‐eye detection and can also be detected measuring electrical conductivity.[98]

Despite the impressive and fast development of this kind of sensors, some obstacles must be circumvented to produce even more suitable viral nucleic acid PoC sensors: the dependence on nucleic acid extraction and amplification. Recent studies show the efforts to improve Cas13 and Cas12‐based detectors. Sabeti’s group implemented heating unextracted diagnostic samples to obliterate nucleases (HUDSON) coupled to SHERLOCK. It enables rapid and sensitive (aM) DNA or RNA detection from body fluids, avoiding the nucleic acid isolation step. It inactivates the viral particles and the RNases in urine, saliva, whole blood, or serum samples with heat and chemical reduction. Those samples can be directly added to RPA reactions.[105] Interestingly, two recent publications show that it is possible to unify the isothermal amplification and target detection steps in one‐pot, preventing cross‐contamination in sample manipulation. The first one, STOPCovid (SHERLOCK testing in one‐pot), uses the thermostable Alicyclobacillus acidophilus (Aap) Cas12b. The system is able to combine viral particles lysis and RNA extraction with one buffer in a short time, followed by RNA concentration using magnetic beads. Then, sample amplification is performed straight from the lysate by RT‐LAMP, and Cas12b detection is performed in one‐pot at 60°C. The use of thermostable nucleases like AapCas12b is then the key point of this kind of methods. Besides, LAMP primers and crRNAs must be carefully designed. Lateral‐flow or fluorescence can be used as readout, allowing to detect aM concentration of SARS‐CoV‐2 N gene, similar to qPCR.[100] The all‐in‐one dual CRISPR‐Cas12a combines RPA isothermal amplification and Cas12a RNP target recognition in one‐pot. It is performed at 37°C, so the better studied LbaCas12a is applied.[101] This method uses two LbaCas12a‐crRNA RNPs, which interact with two different sites within the amplicon, very close to the RPA primers recognition sites. The RPA amplification and the Cas12a‐crRNAs cleavage occur in parallel, together with the trans‐cleavage activity. The system lacks PAM sequence restriction because Cas12a lacks PAM requirements if the target sequence is already unwound.[113] The amplification reaction unzips Cas12a RNP target sites, allowing its PAM‐free interaction with the DNA. Furthermore, the use of two Cas12a RNPs per target increases the specificity. Coupled to a fluorescent read‐out, this method has been used to detect few copies of SARS‐Cov‐2 N gene in clinical samples, presenting aM sensitivity.[113]

Undoubtedly, the most needed development of CRISPR‐based sensors is to extend their detection limit to avoid prior sample isothermal amplification, as this implies an extra‐step that must be optimized for every target. Qin et al.[107] used LwaCas13a, an automated multiplexed microfluidic chip for sample concentration, and a custom integrated benchtop fluorometer to circumvent isothermal amplification. This method detects Ebola virus with 100 fM sensitivity in only 5 min. Although less sensitive than qPCR detection, this work showed the promising future of multidisciplinary approaches to develop better CRISPR‐based sensors. In this regard, English et al. detected unamplified samples combining Cas12a, DNA hydrogels, microfluidics, and electric conductivity detection.[98] Both methods combine microfluidics to increase sample concentration and improve sensitivity.

Recently, Jennifer Doudna and colleagues have developed an LbuCas13a detection system for SARS‐CoV‐2 directly from viral RNA, with no need for previous amplification. They combine various crRNAs carefully designed to improve target recognition. The readout is conducted using a mobile phone camera with laser illumination. The mobile phone works then as a fluorometer, with an app for RNA quantification, allowing for PoC detection. This work shows that sensitivity can be highly increased by just combining various well‐designed crRNAs to recognize the same target. Thus, it should be underlined the importance of the crRNA design step, which can be crucial for effective and sensitive target recognition.[114]

The recent coupling of CRISPR‐based viral sensors with nanotechnology has opened a novel field with great potential. Bao et al. developed a Magnetic Bead‐Quantum Dot (MB‐Qdot)‐based sensing system able to detect nM concentrations of ASFV’s nucleic acids. The system uses streptavidin‐coated MBs and two complementary ssDNA probes tagged with biotin and Qdots, respectively. The biotin probe can bind to MB and interact with the complementary ssDNA labeled with QDot. When the MBs are separated using a magnetic field, the Qdots are pulled down, resulting in a colorless solution. When LbCas12a RNP and its target are previously added to the biotin‐labeled probes, the target‐activated RNP cleaves the biotin probe and the Qdot‐probes cannot bind the MBs, so the solution remains colored after magnetic MB separation.[115] This is a highly portable system due to the naked‐eye readout. Its low sensitivity could be improved by coupling it with prior sample amplification. Hu et al. discovered a fast and efficient AuNPs coating method using poly (A) linkers, based on which they developed a diagnosis system. It relies on the same kind of read‐out than Bao et al. but using AuNPs instead of Qdots. This method was able to detect ASFV from serum samples, after RPA isothermal amplification, although the limit of detection is not provided. They also designed an RNA detection system using AuNPs coated with ssRNA‐FAM oligonucleotides and LbuCas13a. Upon target recognition, the FAM is released and can be detected by fluorescence. This alternative method was used to detect bacteria strains, but not viruses.[102]

Published at Fri, 12 Feb 2021 04:50:39 +0000