CRISPR Systems For COVID-19 Diagnosis

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CRISPR Systems for COVID-19 Diagnosis

Hossein Rahimi, Marziyeh Salehiabar, Murat Barsbay, Mohammadreza Ghaffarlou, Taras Kavetskyy,
Ali Sharafi, Soodabeh Davaran, Subhash C. Chauhan, Hossein Danafar,* Saeed Kaboli,*
Hamed Nosrati,* Murali M. Yallapu,* and João Conde*
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ABSTRACT: The emergence of the new coronavirus 2019 (COVID-19) was first seen in December
2019, which has spread rapidly and become a global pandemic. The number of cases of COVID-19
Downloaded via 177.181.167.104 on November 30, 2022 at 17:06:18 (UTC).
and its associated mortality have raised serious concerns worldwide. Early diagnosis of viral infection
undoubtedly allows rapid intervention, disease management, and substantial control of the rapid
spread of the disease. Currently, the standard approach for COVID-19 diagnosis globally is the RT-
qPCR test; however, the limited access to kits and associated reagents, the need for specialized lab
equipment, and the need for highly skilled personnel has led to a detection slowdown. Recently, the
development of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic
systems has reshaped molecular diagnosis. The benefits of the CRISPR system such as speed,
precision, specificity, strength, efficiency, and versatility have inspired researchers to develop CRISPR-
based diagnostic and therapeutic methods. With the global COVID-19 outbreak, different groups
have begun to design and develop diagnostic and therapeutic programs based on the efficient CRISPR
system. CRISPR-based COVID-19 diagnostic systems have advantages such as a high detection speed
(i.e., 30 min from raw sample to reach a result), high sensitivity and precision, portability, and no need for specialized laboratory
equipment. Here, we review contemporary studies on the detection of COVID-19 based on the CRISPR system.
KEYWORDS: CRISPR, COVID-19, SARS-CoV-2, RT-qPCR, diagnosis
T he emergence of severe acute respiratory syndrome

coronavirus-2 (SARS-CoV-2), also known as the new
coronavirus 2019 (COVID-19), was first seen in Wuhan,
the upper respiratory tract of chickens, while they induce
diarrhea in cows and pigs.6−8 Animal-to-human transmission of
the virus and vice versa was observed with the outbreak of
China in December 2019. This devastating disease has caused acute respiratory syndrome (ARS) and MERS in 2002/2003
a huge number of deaths worldwide, and the death toll is and 2012, respectively.9,10
increasing by the day. Coronaviruses, a large human and The positive-sense RNA genome of coronaviruses is 26 to
animal pathogen, are a group of enveloped viruses and have an 32 kb in size and has a variable number of open reading frames
RNA genome. Because of the crown-like structure on their (ORFs) ranging from 6 to 11. The first ORF, which encodes
surface, these viruses are called coronaviruses (in Latin, 16 nonstructural proteins, represents approximately 67% of the
“corona” means “halo” or “crown”). This group of viruses entire genome, while accessory and structural proteins are
was first identified in chickens1 and humans2,3 in the 1930s and encoded by other ORFs.9,11 Structural proteins encoded by the
1960s, respectively. These viruses constitute the largest group genome of these viruses include spike (S), nucleocapsid
in the order of the Nidovirales. Coronaviridae, Arteriviridae, protein (N), membrane (M), small envelope protein (E), and
Mesoniviridae, and Roniviridae are the families of the hemagglutinin-esterase glycoprotein (HE; Figure 1I).12 S
Nidovirales. The family of Coronaviridae is classified into proteins are virus-binding mediators to host receptors through
two groups, Coronavirinae and Torovirinae. The Coronavir- domains that bind to the receptor.7 M protein, with an
inae subfamily is composed of alpha, beta, gamma, and delta approximate molecular weight of 25−30 kDa, is the virion’s
groups which infect humans by alpha (229E, NL63) and beta most abundant structural protein and is involved in defining
(OC43, HKU1) groups. Middle east respiratory syndrome
(MERS) and severe acute respiratory syndrome (SARS)
coronaviruses are other human beta-group coronaviruses.4,5 Received: November 4, 2020
Coronaviruses in humans can cause infections in respiratory, Accepted: January 18, 2021
gastrointestinal, and liver systems, leading to both mild Published: January 27, 2021
symptoms such as colds and fatalities such as MERS.
Moreover, the symptoms caused by these viruses are different
in other animal species; for example, they cause infections in
© 2021 American Chemical Society https://dx.doi.org/10.1021/acssensors.0c02312

1430 ACS Sens. 2021, 6, 1430−1445
ACS Sensors pubs.acs.org/acssensors Review
Figure 1. Schematic representation of various diagnosis tools for SARS-CoV-2. I. Types of methods used to diagnose SARS-CoV-2 and II. SARS-
CoV-2 diagnostic methods results’ readout ways.
the shape of the viral envelope.13 The M and E proteins form a become currently a powerful genome editing system, relies on
viral envelope together and interact to produce and release RNA-guided activity of Cas proteins.23 In addition to genome
viral particles.4,14 N protein is the only protein that binds to editing, CRISPR/Cas technology has recently gained a lot of
the RNA genome of the virus and triggering nucleocapsid interest in the area of nucleic acid detection owing to its
formation.15 unique features. To date, different variants of the CRISPR
Early diagnosis of a viral infection may allow for rapid systems have been used to design and develop simple, portable,
intervention, which can effectively minimize the risk of precise, efficient, rapid, and inexpensive molecular detection
spreading the disease to others. PCR is one of the most methods. In general, there are two major parts in the CRISPR
common techniques used to detect viral nucleic acids and, due system: Cas endonuclease (to break the target genomic site)
to its high sensitivity and accuracy, is used as the gold standard and guide RNA (to identify and direct Cas endonuclease to the
technique for diagnosing viral infections. The quantitative PCR target region).24 Successful detection of nucleic acids by the
(qPCR) test can be used to diagnose COVID-19, but limited
dead Cas9 (dCas9) system (is a mutant form of Cas9 whose
access to qPCR equipment and materials may slow down the
endonuclease activity is removed through point mutations in
diagnosis process.16−19 Loop-mediated isothermal amplifica-
its endonuclease domains) has been reported several
tion (LAMP) and recombinase polymerase amplification
(RPA) are approaches that have emerged as effective and times.25,26 Cas12 and Cas13 endonucleases have collateral
appealing alternatives to PCR due to their cheapness, high cleavage activities that can be used to detect nucleic acid. For
speed, and versatility.20,21 The isothermal amplification feature, example, cutting a fluorescent reporter using the collateral
which is performed at a constant temperature and does not cleavage activity of these endonucleases can produce a
require thermal cycling, is one of the key aspects of RPA and fluorescent signal that results in diagnosis. In addition, Cas9
LAMP and can also be performed with minimal tools and endonuclease specific cleavage activities have been used to
equipment.22 CRISPR, discovered in the 1980s and eventually establish highly sensitive and accurate diagnostic tools for
becoming a tool for genome editing, is an adaptive immune DNA detection. RPA and LAMP isothermal amplification
system in prokaryotic organisms such as bacteria and archaea approaches are typically used in CRISPR-based diagnostic
that protects the organism against foreign and invasive genetic works to amplify target genomic sequences.27 However, in an
elements such as viruses and plasmids. This system, which has attempt, a Cas13a-based diagnostic system was developed to
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detect SARS-CoV-2 that does not require preamplification of important limiting factors for molecular diagnosis is detection
the virus genome.28 time.40,41
■
There have been significant advances in the molecular
DETECTION OF SARS-COV-2 BASED ON detection of COVID-19 since its outbreak. Although testing
MOLECULAR TECHNIQUES capability cannot currently address the global need for rapid
COVID-19 diagnosis, fundamental issues such as false-negative
Efficient detection of foreign genomes and pathogens in results and the development of faster, highly sensitive, and
clinical specimens, as well as changes in their sequences, are affordable diagnostic procedures remain to be tackled. Delays
critical to the diagnosis and management of specific diseases. in patient care, as well as an increased risk of asymptomatic
Early and efficient detection of SARS-CoV-2 was first spread to others, are concerns related to the false-negative
established by analysis of pathogenic sequences in respiratory results in SARS-CoV-2 patients. False negative results can be
tract samples, which is important to limit transmission of the caused by factors such as sampling and sample analysis.42
virus and maintain disease control.29 A variety of methods Depending on the time of infection and the place where the
including RT-qPCR, sequencing-based methods, nanotechnol- sample was taken, the viral load in a sample can be vary. For
ogy-based assays, immunological assays, and the CRISPR/Cas instance, following the appearance of illness, the viral load
system have been used to diagnose SARS-CoV-2 (Figure 1I) varies depending on the timing of collection between the nasal
and along with appropriate readout (Figure 1II) were shown in and oral swabs.43 During the progression of the disease, the
Figure 1. The medical nanotechnology revolution has opened a variation in viral load at different locations makes sampling
window of hope for the treatment and diagnosis of a wide more challenging and allows the production of false-negative
range of diseases, especially cancer and viral diseases. Owing to results. Some other factors, such as mistakes in the collection
the unique properties of nanomaterials and the effectiveness of of clinical samples, degradation of RNA, inefficient extraction
medical nanotechnology against various infectious diseases of RNA, and insufficient purification of RNA, may contribute
such as HIV-1, influenza virus, HBV, and respiratory syncytial to false negative results. Positive RT-PCR test results
virus, researchers are urged to develop nanotechnology-based demonstrate SARS-CoV-2infection, but negative RT-PCR
tools for SARS-CoV-2 disease management.30,31 Rapid and results do not reject SARS-CoV-2 infection. Molecular
specific diagnosis of SARS-CoV-2, targeted delivery of antiviral diagnosis should be used in combination with other diagnostic
agents to different parts of the body, and preparation of information such as patient medical history, clinical observa-
nanobased disinfectants are some of the applications of tions, and epidemiological surveillance information to ensure
nanotechnology in the management of SARS-CoV-2 disease.32 the diagnosis of the disease.42 The most common methods
Immunological tests can detect the presence of COVID-19 used in the clinic to detect foreign gene material include qPCR,
antivirus antibodies or viral antigens (viral structural proteins). next generation sequencing (NGS), and fluorescence in situ
Enzyme linked immunosorbent assays (ELISAs), chemilumi- hybridization (FISH), which have changed the landscape of
nescence immunoassays (CLIAs), and lateral flow immuno- molecular detection. Despite fundamental improvements, the
assays (LFIAs) are immunological methods that have recently use of such methods still presents challenges. While the qPCR
been used to diagnose SARS-CoV-2.33 While diagnostic tests test is the primary tool utilized in clinical laboratories to
based on nucleic acid amplification are suitable for early stage recognize the causative agent of the CoVID-19 (e.g., SARS-
diagnosis of viral diseases, immunological diagnostic tests allow CoV-2), it may face problems such as the need for highly
the diagnosis of a past or ongoing infection, which provides a trained technicians, advanced thermocyclers, and well-
better understanding of the mechanism and dynamics of equipped laboratories.44−46
disease transmission.34 In the diagnosis of COVID-19, One of the valuable molecular tools used to study the
immunological diagnostic tests mainly target the structural function and regulation of genes, and specifically viral
proteins of the virus. Since the S protein is the major detection, is the in situ hybridization (ISH) technique. This
transmembrane protein of SARS-CoV-2 and is highly technique uses hybridization probes (made of single- or
immunogenic, this protein may be a very suitable option as double-stranded nucleic acids or synthetic oligonucleotides) to
an antigen in the diagnosis of SARS-Cov-2.34,35 In addition, detect and localize a specific nucleic acid in a tissue sample or
the receptor-binding domain (RBD) located along the cell. By attaching a reporter to the complementary strand of
structural S protein is a target for the detection of COVID- the desired nucleic acid in a known labeling method,
19-specific antibodies.34 hybridization probes are produced. If fluorescence is used in
One of the main ways to diagnose COVID-19 infection is to the ISH technique, this technique is called FISH.47,48 The
identify the SARS-CoV-2 RNA genome.36,37 While molecular FISH approach also offers a single-cell system to analyze the
diagnostics can be developed rapidly and provide very high number of copies, replication, and gene rearrangement that
precision and sensitivity and also quantitative detection of may influence targeted therapy. The requirement for long-term
SARS-CoV-2 nucleic acid, it still faces issues such as high cost, high-temperature treatment to hybridize the FISH probe is
difficulty, and the need for highly skilled technicians and time-consuming, as well as cell morphology weakening, which
equipped centers. For example, an RT-PCR kit can cost more contributes to the possibility of missing spatial structure
than $100 U.S., and sample analysis can take about 4 to 6 h, information. Additionally, toxicity caused by formamide used
and more than 24 h from raw sample to the final result may be for denaturation of double-stranded DNA is another obstacle
required. Information on the amount of detectable viral titer in in the FISH procedure.49 This technique also faces challenges
the respiratory tract at various stages of SARS-CoV-2 infection such as false positive and negative results, incomplete
is still expanding. The kinetics of viral load can also differ from hybridization, processing problems, nonspecific binding, and
person to person and depend on various factors such as the photobleaching.48
patient’s epidemiological history, immune response, and effects The massively parallel sequencing technology, known as
of medication and treatment.38,39 Hence, one of the most next generation sequencing (NGS), has revolutionized
ACS Sens. 2021, 6, 1430−1445
molecular biology. The benefits of NGS technology including

ultrahigh throughput, speed, and scalability have allowed
researchers to conduct a broad variety of biological studies that
were not feasible until this technology was developed. This
technology can detect all kinds of nucleic acid sequences in a
single sample, so that NGS can recognize all known viruses as
well as new ones that have not yet been detected.50
Nevertheless, the widespread use of NGS is constrained by
concerns such as insufficient target enrichment, which raises
costs as well as reduces sensitivity.51,52 In reality, the biggest
obstacles to converting NGS technology into a routine
diagnostic test are the high cost and high processing time of
achieving the final result from the raw sample. Compared to
RT-PCR, NGS technology has a high cost and high
turnaround time. Therefore, the difficulty and time-consuming
nature of NGS-related methods restricts the usage of NGS
when rapid results are required.53
■ CRISPR/CAS-BASED SYSTEMS FOR COVID-19

DETECTION
The CRISPR system is a simple, efficient, and reliable system
that enables researchers to make desired changes in genomic
sequences that may alter gene function. This system, which Figure 2. Schematic illustration of SHERLOCK and DETECTR
workflow and the main mechanisms involved in CRISPR-based
functions like a pair of molecular scissors and can cut DNA
diagnosis systems.
strands, is a family of DNA sequences found in prokaryotes
such as bacteria and archaea.54−57 Generally, the CRISPR
system is divided into two main classes and six types. detection systems may become widely available and replace
The first class includes type I, III, and IV systems, while the conventional methods such as PCR. The application of
second class includes type II, V, and VI systems. In class I, type CRISPR-based diagnosis systems in the molecular diagnostic
I Cas3 nuclease is used to cut DNA, and type III Cas10 field has grown, and a variety of CRISPR-based diagnostic
nuclease can cut RNA. In class II, type II systems use Cas9 tools for diagnosing infectious and noninfectious diseases have
endonuclease to cut DNA, and type V uses Cas12 to cut DNA. been created so far. Here, we review all the studies conducted
In addition, Cas13 nuclease is used in class II, type VI systems to diagnose SARS-CoV-2 infection using the CRISPR/Cas
to make cuts in target RNA. The CRISPR system functions as system (Cas12, Cas13, Cas9, and Cas3; Figure 3).71−74
the bacteria and archaea’s adaptive immune system against Table 1 summarizes studies on the development and design
foreign elements including viruses or plasmids.58−62 Class II of CRISPR-based diagnostic systems for rapid and sensitive
diagnosis of COVID-19.
■
CRISPR systems are used widely for genomic manipulation
and infectious disease diagnosis. For example, CRISPR/
Cas12a, CRISPR/Cas13a, and CRISPR/Cas13b systems have DIAGNOSIS OF SARS-COV-2 BASED ON
been used in recent years to develop rapid and sensitive CRISPR/CAS12
diagnostic methods for human pathogen (bacteria and viruses) Broughton et al.16 developed a CRISPR/Cas12a-based
detection.63−67 A multiplex diagnostic system developed by precision technique, called SARS-CoV-2 DNA endonuclease-
Kellner et al.68 recently incorporated nucleic acid preamplifi- targeted CRISPR trans reporter (DETECTR), which allows
cation with CRISPR/Cas enzymology to accurately identify for the simple and rapid diagnosis of SARS-CoV-2 RNA
the targeted nucleic acid sequences. The developed system, extracted from patient respiratory tract swab samples in less
called specific high-sensitivity enzymatic reporter unlocking than 40 min. Actually, the developed method is the product of
(SHERLOCK; Figure 2), can detect clinical sample nucleic combining the CRISPR/Cas12a DETECTR system with
acid sequences in a portable and ultrasensitive manner.68 DNA isothermal amplification that simultaneously performs reverse
endonuclease-targeted CRISPR trans reporter (DETECTR; transcription and isothermal amplification by loop-mediated
Figure 2) is another CRISPR-based diagnostic system replication (RT-LAMP) for purified RNA from nasophar-
(CRISPR/Cas12) that detects viral infections rapidly (∼30 yngeal or oropharyngeal swabs. Cas12a then detects
min), inexpensively, and accurately.60 The two aforementioned predetermined viral sequences, whereupon the reporter
diagnostic systems (SHERLOCK and DETECTR), which molecule’s cleavage confirms the presence of a virus.
have high specificity and sensitivity, are comparable to Eliminating the need for thermocycling and isothermal signal
conventional diagnostic methods such as PCR but do not amplification offers significant benefits compared to qRT-PCR,
require expensive advanced equipment.60,68 To the best of our such as fast turnaround time, target specificity for single
knowledge, DETECTR and SHERLOCK diagnostic kits for nucleotides, integration with usable and user-friendly reporting
SARS-CoV-2 detection have been approved and are formats like lateral flow strips, and no requirement for
commercially available. sophisticated laboratory systems. The established system was
PCR and other conventional approaches for amplifying validated with reference samples and also United States patient
DNA or RNA sequences for recognition are reliable but still samples including 36 patients with COVID-19 and 42 patients
demand nonportable tools that prevent their spread in the area with other respiratory viral infections. This system is a high-
of diagnosis.69,70 Thus, in the near future, CRISPR based speed and visual alternative to rRT-PCR for detecting SARS-
ACS Sens. 2021, 6, 1430−1445
Figure 3. Schematic illustration of the most commonly used CRISPR systems, CRISPR-Cas (Cas3, Cas9, Cas12, and Cas13), for detection of
SARS-CoV-2.
CoV-2 with 95% and 100% agreement for positive and samples of SARS-CoV-2 cases in this study may be criticized,
negative prediction, respectively.16 but no real samples were used in this study due to the lack of
The all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) reports of patients suffering from SARS-CoV-2 disease in the
system was developed by Ding et al.75 for faster, highly study area (South America). To mimic real samples, synthetic
sensitive, highly specific and optical-based nucleic acid SARS-CoV-2 nucleic acid sequences were added to a healthy
detection. This method uses dual crRNAs to efficiently detect person’s saliva sample. The use of saliva to diagnose SARS-
the target genome sequence. The AIOD-CRISPR system CoV-2 is a reasonable approach as it is completely noninvasive
integrates all the components needed for target nucleic acid and sampling is quick and easy. It was found that the CRISPR
amplification as well as CRISPR system-based detection into a diagnostic system used in this research is not inactivated in
single reaction that resulted in deletion of the need for separate saliva, so it is a promising tool for accurate and fast identify of
amplification and transfer of amplified product. The AIOD- real SARS-CoV-2 samples.76
CRISPR system was designed to be used for the detection of Wang et al.77 developed a rapid and accurate CRISPR/
SARS-CoV-2 and HIV-1. As both viral agents are retroviruses, Cas12a-based system that allows reading with the naked eye
the capability of the AIOD-CRISPR system to detect each of (CRISPR/Cas12a-NER) to boost and accelerate the detection
their nucleic acid states (DNA and RNA) was assessed and of the SARS-COV-2 Genome.77 CRISPR/Cas12a-NER can
their nucleic acids were detected successfully.75 rapidly, reliably, and sensitively detect at least 10 copies of a
Among the various classes of CRISPR systems, the viral gene in 40 min without the demand for specialized
CRISPR/Cas12 system, an RNA-guided DNase, causes instruments. The designed system consists of Cas12 protein,
single-stranded DNA cleavage after identifying the target SARS-COV-2-specific crRNAs, and a single-stranded DNA
region. The CRISPR/Cas12 ability to create collateral cleavage molecule as a reporter (labeled with a green fluorescent off−on
in single-stranded DNA can be enjoyed to destroy single- molecule). Where the genome of SARS-COV-2 is present in
stranded reporter molecules that generate fluorescent signals.65 the sample and detected by the designed diagnostic system, the
Thus, CRISPR-Cas12-related approaches can be used in real reporter molecule is cleaved by the Cas12 protein, resulting in
time as an in situ diagnostic tool for diagnosing SARS-CoV-2 green fluorescent light visible to the naked eye at 458 nm.77
infection. The CRISPR/Cas12a system was implemented in Inexpensive, highly sensitive, precise, high-throughput, and
another attempt to identify the synthetic SARS-CoV-2 nucleic highly effective methods are needed to control the disease and
acid sequences reliably, sensitively, and in a rapid manner.88 recognize even asymptomatic patients in any region. Studies
Synthetic sequences of the RdRp, ORF1b, and ORF1ab genes have shown that, although SARS-COV-2 patients have no signs
were considered as references in this work. The lack of actual or presymptoms, they are strongly contagious and can infect
ACS Sens. 2021, 6, 1430−1445
Table 1. Rapid and Sensitive Detection of COVID-19 with CRISPR-Based Diagnostic Platforms
system Cas type test timing advantages shortcomings ref
DETECTR Cas12a 30−40 accurate, easy-to-implement, rapid turnaround time, no need for thermocycling, single nucleotide target specificity, and needs for nucleic acid extraction, limited access to 16
min no need for complex laboratory infrastructure extraction, kits and reagents, needs for personal
ACS Sensors
protective equipment
AIOD- Cas12a 40 min rapid, highly sensitive, highly specific, one-pot reaction, no need for separate preamplification and amplified product needs for nucleic acid extraction, limited access to 75
CRISPR transferring, visibility of results with the naked eye, nucleic acid detection in both DNA and RNA states, performable in extraction, kits and reagents
one-step, single-molecule sensitive, and robust
CRISPR- Cas12a Less than portable, sensitive, rapid, and low cost patient samples are not used and requires certain 76
Cas12 based 60 min kits
CRISPR/ Cas12a 45 min portable, simple, sensitive, specific, no need for special instrument, rapid, and visibility of results with the naked eye needs for nucleic acid extraction, limited access to 77
Cas12a-NER extraction, kits and reagents
CRISPR-FDS Cas12a ∼50 min sensitive, robust, rapid, and can be done with available equipment needs for nucleic acid extraction, not suitable for 78
quantifying viral load
SHINE Cas13a 50 min sensitive, specific, single-step reaction, can be used outside of hospitals and laboratories, and no need for nucleic acid 79
extraction
CONAN Cas3 40 min rapid, sensitive, low-cost, instrument-free, and single-base-pair discrimination needs for nucleic acid extraction, limited access to 80
extraction, kits and reagents
iSCAN Cas12a 1h sensitive, specific, efficient, rapid, user-friendly, accurate, field-deployable, and suitable for large-scale requires nucleic acid extraction, limited access to 81
CASdetec Cas12b 1h no cross-reactivity, reduced false positive rate, and accuracy needs for nucleic acid extraction, limited access to 82
VaNGuard Cas12a 30 min robust, rapid, sensitive, affordable, specific 83
CREST Cas13a ∼2 h scalable, low-cost, no need for specialized instrumentation, highly sensitive, easy to deploy requires nucleic acid extraction, limited access to 84
1435
STOPCovid Cas12b 1h simple, suitable for point-of-care (POC) analysis, sensitive, low-cost, availability of test components, no need for RNA 85
extraction
ITP-CRISPR Cas12a 30 min amenable to automation and the use of a minimum volume of reagents 86
SHERLOCK Cas13a less than rapid, sensitive, and no need for sophisticated equipment not fit to test clinical samples 87
1h
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ACS Sens. 2021, 6, 1430−1445

https://dx.doi.org/10.1021/acssensors.0c02312
many others. Therefore, these conditions encourage research- DNA detection (CDetection) was established for the detection
ers to establish fast, precise, and highly sensitive methods for of SARS-CoV-2 in an attempt by Guo et al.82 By incorporating
SARS-COV-2 detection on a large-scale. In an attempt to meet sample treatment protocols and nucleic acid amplification
these demands, a diagnostic system based on CRISPR was strategies with CDetection, they set up an integrated viral
developed by Huang et al.78 The method consists of CRISPR/ nucleic acid detection systemCASdetect (CRISPR-assisted
Cas12a system and a fluorescent probe to diagnosis RT-PCR detection). The CASdetect system’s limit of detection was 1 ×
or RPA amplified amplicons, allowing sensitive and accurate 104 copies/mL for the identification of the SARS-CoV-2
detection in areas that do not have a real-time PCR system. It pseudovirus, without cross-reactivity to other human endemic
was observed that the developed system was capable of coronaviruses.82
detecting positive SARS-COV-2 samples in about 50 min, with Nucleic acid amplification based molecular detection is
the limitation of detecting two copies of the target RNA currently the most reliable, fast, and inexpensive approach for
sequences for each sample. However, in samples with less than SARS-CoV-2 disease diagnosis; quite a few companies and
five copies of the amplified target DNA, the qPCR test failed to laboratories have developed rRT-PCR kits.90 The rRT-PCR
produce a detectable signal. The use of CRISPR in detection performance for SARS-CoV-2 detection presents a number of
has become very common due to the ability of Cas13 to bind urgent challenges particularly with uncertain negative or
to the target RNA molecule and Cas12 to bind to the target positive results associated with the frequently encountered
DNA molecule by the gRNA sequence to make a cleavage in “gray zone” designated with high Ct value.91−95 In addition to
the probe to produce the detection signal.60,67 Most CRISPR- user errors such as inaccurate sampling, poor quality reagents,
based diagnostic methods use paper strips to detect the output and uncalibrated instruments, ineffective RT reaction and PCR
signal. Using paper strips to recognize single samples is a proliferation of patient specimens with very poor virus titers
reasonable strategy since the tests do not require special are probably main causes of inaccurate rRT-PCR readouts and
equipment, but the limit of detection (LoD) value obtained is unclear diagnosis. While the diagnosis can be verified through
very low compared to fluorescence-based approaches. The repeated sampling and control, troubleshooting efforts are
CRISPR-FDS method developed by Huang et al.78 could be time-consuming, and low-viral load specimens will not be
conveniently implemented on 96-well microtiter plates that detected in mild or asymptomatic patients or in advanced
can be used in most well equipped laboratories with precision cases, therefore this leads to a false negative result that may
and ease using fluorescent plate readers.78 Overall, the results raise concerns about fighting the disease.96 The identification
of this study were comparable to the results of the RT-qPCR of nonspecific trans-cleavage activities in various Cas proteins,
test performed in public laboratories; however, the CRISPR- including Cas12, Cas13, and Cas14, contributed to the rise of
FDS method provided more reliable results than those the CRISPR-Diagnostics strategy (CRISPR-Dx).60,66,67,97−102
reported in a clinical setting when the same RT-qPCR assay The mechanism of action of the CRISPR-Dx technique is
was used. It should be stated that the CRISPR-FDS method based, as demonstrated by the Cas12a-based HOLMES
has provided positive results for certain samples that have system,103 on the efficient trans-cleavage activity of Cas12a
obtained negative results using the RT-qPCR test, although it (upon detection of target DNA) against single-stranded DNA
is not possible to decide if these results are false positives for labeled with a fluorophore quencher (FQ), whose fluorescence
CRISPR-FDS or false negatives for RT-qPCR owing to a signal increases exponentially within a few minutes. On the
shortage of valid follow-up data. Therefore, the CRISPR-FDS basis of this mechanism, Huang et al.96 developed a specific
method can be considered as a sensitive and powerful method enhancer for the identification of nucleic acids amplified by
for producing results using tools and available equipment for PCR (SENA) to improve the accuracy and efficiency of
use in clinical laboratories as well as care facilities with detecting preamplified nucleic acid sequences of SARS-CoV-2.
appropriate equipment. The CRISPR/FDS system is designed In summary, they first analyzed SARS-CoV-2 samples using
so that it produces clear negative or positive results and cannot rRT-PCR and then validated amplicons with unclear readouts
quantify virus titers like RT-qPCR, which is a drawback for the by SENA.96 The SENA diagnostic system was highly sensitive
CRISPR/FDS system. However, given that the main goal is to and specific and was able to detect false positives and negatives
develop a quick, accurate, and reliable COVID-19 detection with a detection limit of two copies per reaction less than the
system, this deficiency cannot be considered an important corresponding rRT-PCR test. Initially, the amplicon sequences
flaw.78 were determined from different rRT-PCR kits to design
A diagnostic method consisting of a combination of suitable cRNAs for the SENA, and then a specific crRNA was
CRISPR/Cas12a and RT-LAMP, called the in vitro specific designed for each amplicon. Then, candidate crRNAs were
CRISPR-based assay for nucleic acid diagnosis (iSCAN) screened on the SENA consisting of Cas12a, crRNA, FQ-
system, was introduced in an attempt by Ali et al.89 to reporter, and rRT-PCR products, and the best crRNAs were
overcome the shortcomings of SARS-CoV-2 detection.89 The selected for further SENA testing.
potential benefits of the iSCAN system include: (i) high speed, Owing to the sampling distribution of Poisson, replica
(ii) precision (because of the diagnosis reliance on SARS-CoV- variations are very important when copies of templates are
2 nucleic acid sequences being detected and cut by Cas12), designed to be small (below 3−4 copies/Rx), close to the LoD
(iii) field-deployability (since it needs only the basic tools), for rRT-PCR, and very low (equal to and less than 1 copy/
and (iv) easy operation (since it provides easy access to test Rx).104,105 To address the sampling issue, nine replicas were
results by incorporating a colorimetric reaction with lateral conducted for groups of RNA templates with a half and one
immunochromatography flow). The iSCAN system is well copy/Rx, while six replicas were conducted in each of the other
suited for early identification of COVID-19 carriers so that concentration levels. Following the rRT-PCR reaction, all
they can be identified and quarantined early, thereby amplicons were entered into three different SENA reactions
preventing the virus from spreading widely.89 Another with crRNAs related to O and N genes and both (N-SENA, O-
diagnostic tool based on CRISPR called Cas12b-mediated SENA, and mix-SENA). By reducing RNA templates to less
ACS Sens. 2021, 6, 1430−1445
than three copies per reaction, it was found that Ct values gradient could be obtained using isotachophoresis, a special
exceeded 38 (cutoff for positive) in some replicas mainly ionic focusing technique, applied to a microfluidic chip.
related to the N gene, but when it drops below 40, it is Overall, on-chip electric field control and microfluidics were
considered to enter the gray zone. As the concentration of combined to perform two very important steps: (1) automatic
RNA templates decreases, Ct values increase steadily, with extraction of nucleic acid from primary biological samples
most replicas indicating that one or both Ct values enter the (here, nasopharyngeal samples of patients with COVID-19 and
gray zone, and ultimately, they all become negative. Taking Ct healthy controls were used) and (2) application of an electric
= 38 as the cutoff for positive detection, the detection limit for field to monitor and influence the rapid enzymatic activity of
O and N genes with a 95% confidence interval (CI) of this set the CRISPR/Cas12 complex as soon as the target nucleic acid
of rRT-PCR was estimated to be 3.3 ≤ 4.0 ≤ 6.1 and 4.0 ≤ 4.1 sequence is detected. This allows simultaneous combination of
≤ 4.4, respectively. The rRT-PCR amplicons were then enzymatic reactions, preconcentration, and speed. This
examined with the SENA system to measure the signals of developed microfluidic system not only needs a small volume
fluorescence for each replica and after comparing the increase of reagents but also is open to automation. It was assessed for
in fluorescence versus the rate of fluorescence change between detecting SARS-CoV-2 nucleic acid in COVID-19 positive and
samples at a given time relative to the negative control (FC), healthy samples. Ultimately, as a remarkable difference
the FC parameter was defined as the ratio of FC values compared to current COVID-19 test procedures, this process
measured at the 10th and fifth minutes following the takes just 30 min from the raw sample to be completed.107
fluorescence reading starting. It has also been found that the As discussed above, Cas12 has been used for the detection
effectiveness of rRT-PCR for both O and N genes is different of SARS-CoV-2 in three states. Cas12 is able to detect the
from that of the SENA system with decreasing template SARS-CoV-2 genome both before and after nucleic acid
concentration. The amplicons of the RNA template were extraction, which schematically is shown in Figure 4.
investigated via NGS, and it was found that the findings were
found to be consistent with O-SENA and mix-SENA results. In
addition to avoiding false-positive and false-negative diagnoses,
the highly sensitive SENA system can be useful in
demonstrating that the virus has been cleared from recovered
cases. Since the qPCR system is still the most popular
molecular detection system, and the SENA system is
remarkable for operational simplicity, the SENA system can
be widely used to solve fuzziness troubles of qPCR and other
molecular detection systems based on amplification of nucleic
acids.96
Recent trends in effective CRISPR-based diagnostic systems
demonstrate that the DTECTR diagnostic system can be
applied as a convenient, inexpensive, and rapid substitute to
qRT-PCR, avoiding the loss of sensitivity and specificity for
molecular detection. A comparison of the DETECTR system
with qRT-PCR for SARS-CoV-2 diagnosis in 378 patients
revealed a 95% consensus in an attempt by Brandsma et al.106
Clinical sample dilution assessments showed DETECTR’s
higher analytical sensitivity compared with qRT-PCR, but it
was not verified in a majority of patients. The findings revealed
that both DETECTR and qRT-PCR techniques were similarly
sensitive for the SARS-CoV-2 diagnosis. In the DETECTR
system, different gRNAs could be used simultaneously to
obviate negative results owing to N gene mutations. The
DETECTR system was 100% specific to detecting nucleic acid
sequences of SARS-CoV-2 and therefore could not recognize
Figure 4. Overview of CRISPR/Cas12 based systems used for SARS-
other human coronaviruses. In addition, since the DETECTR CoV-2 detection.
system for SARS-CoV-2 diagnosis does not require specialized
■
equipment and can be used as an independent qRT-PCR
method in diagnostic laboratories, PCR systems in the
laboratory can be used for other diagnostic works.106 SARS-COV-2 DIAGNOSIS BASED ON
CRISPR-based diagnostic systems are currently considered CRISPR/CAS13
as field-deployable solutions. The CRISPR-Cas12/gRNA The CRISPR/Cas13 based diagnostic system’s performance
complex is a basic form of such systems. Cas12/gRNA is depends on the base pairing between crRNA and the target
activated when it specifically binds through gRNA to the target sequence, making such a system a highly accurate and
DNA sequence and then nonspecifically cleaves the programmable diagnostic tool.66,108 Although such systems
fluorophore-quencher pair-labeled single-stranded DNA re- have a high level of sensitivity and diagnostic efficiency, they
porter probe. It has recently been shown that electric field require nucleic acid extraction, which is often restricted in
gradients can be used to control and accelerate this CRISPR- terms of access to the extraction kits, as well as multiple sample
based diagnostic system by cofocusing the Cas12/gRNA transfer steps that hinder ease of use. To eliminate the need for
complex, reporter, and target.88 An appropriate electric field nucleic acid extraction, Myhrvold et al.65 merged the heating
ACS Sens. 2021, 6, 1430−1445
unextracted diagnostic samples to obliterate nucleases that affect the CRISPR/Cas system’s ability to recognize the
(HUDSON) system with the SHERLOCK system. The target area. It has been shown recently that when SARS-CoV-2
developed system employs chemical reduction and heat to enters the body its RNA genome is edited by the deaminases
the degradation of nucleases that break down RNA and lysis including ADAR and APOBEC, which are part of the human
viral particles.65 Overall, it is practical to use the combined immune system against viral attacks.88 Changes in nucleotides
SHERLOCK and HUDSON system in laboratories with during conversions of adenosine-to-inosine and cytosine-to-
limited facilities, as only one heating system is needed. uracil can also affect the ability of the CRISPR/Cas system to
However, its scalability has been limited due to the need to recognize the virus.
prepare multiple reaction mixtures and transfer samples Therefore, the variant nucleotide guard procedure was
between them. A fast and sensitive system called SHINE developed to overcome these challenges and increase the
(SHERLOCK and HUDSON integration to navigate epi- capacity and strength of the diagnostic system for specific and
demics) that did not require extraction of nucleic acid was sensitive detection, as well as to identify mutated and altered
established in a worthy attempt to detect the SARS-CoV-2 nucleic acid regions of SARS-CoV-2.83 The DETECTR system
nucleic acid. Following the development of a SARS-CoV-2 and different forms of Cas12a enzymes were initially studied,
test,109 diagnostic steps based on the Cas13a system and and it was found that enAsCas12a had the greatest tolerance in
SHERLOCK’s amplification were combined, which shortened the CRISPR target area for single mismatches. Interestingly,
both the testing time and operator interventions. The SHINE enAsCas12a was highly specific to SARS-CoV-2 nucleic acid
system has been shown to be able to detect the SARS-CoV-2 and also demonstrated no cross-reactivity with SARS-CoV and
genome in clinical samples treated with HUDSON in paper- MERS-CoV, two other related coronaviruses. Additionally,
based colorimetric or in-tube fluorescent readout methods that various gRNAs were investigated, and all nucleases tested,
can be applied using portable devices and with a low except enRR, were found to show that S2 gRNA leads to
probability of contamination of samples. To evaluate the higher trans-cleavage activity. Thus, the enCas12a-S2 gRNA
performance of the SHINE system, 50 nasopharyngeal has proven to be a powerful and very sensitive system for
specimens (30 RT-qPCR-confirmed SARS-CoV-2 positive detecting SARS-CoV-2 compared to LbCas12a-N-Mam
specimens and 20 SARS-CoV-2 negative specimens) were gRNA. Remarkably, this method can be applied using a
examined. The SHINE system was evaluated on six positive dipstick within 30 min.83
COVID-19 samples using the paper-based colorimetric A protocol called Cas13a-based, rugged, equitable, scalable
method, resulting in the SARS-CoV-2 nucleic acid being testing (CREST) was developed by Rauch et al.84 to overcome
identified in all six positive samples and not in negative control the scalable testing challenges of SARS-CoV-2 diagnosis, such
samples, indicating 100% concordance. The use of the SHINE as access to tools and materials, highly skilled operators, and
system to test 50 samples using the in-tube fluorescence investors. Based on CRISPR/Cas13a, this diagnostic protocol
method resulted in SARS-CoV-2 nucleic acid identification in uses readily available reagents and equipment and is also highly
27 out of 30 positive samples of SARS-CoV-2 and no false sensitive to the detection of SARS-CoV-2. The CREST
negative results within a sample-to-result period of 50 min, technique utilizes the sensitivity and convenience of a
indicating significantly higher specificity (100%) and sensitivity transcription-recognition reaction and also benefits from the
(90%) in comparison with RT-qPCR.79 PCR method’s robustness and reliability. With management in
Metsky et al.109 developed a series of test designs and ∼2 h, without the need for AC power or a sophisticated
experimental procedures for use in the diagnostic systems that facility, CREST can be carried out from an RNA sample to a
focused on CRISPR and could be useful for continuous result.84
surveillance. The presented designs were developed to detect New alternatives to RT-qPCR have decreased RT-qPCR
67 species and subspecies of viruses, such as SARS-CoV-2. The dependency for the detection of SARS-CoV-2, which focuses
algorithms developed by Metsky et al.109 allow molecular on the combination of isothermal amplification and CRISPR
detection assay designs, the output of which is also promising systems, such as the SHERLOCK system. With this in mind, a
for improvement of diagnostics for the detection of viral simple chemical method called SHERLOCK testing in one pot
species. On the basis of the CRISPR/Cas13a (SHERLOCK) (STOP) was established in a valuable effort to diagnose SARS-
system, they screened four of the existing designs for SARS- CoV-2, which is suitable for use at the point of care and can be
CoV-2 diagnosis and then thoroughly evaluated the best carried out within an hour. The STOPCovid method is
performing SARS-CoV-2 assays. This assay was used to comparable in sensitivity to RT-qPCR-based techniques and
demonstrate the sensitivity of SARS-CoV-2 synthetic sequen- also has an LoD of 100 copies per reaction of the viral genome
ces, which was 10 copies per microliter.109 in the samples of saliva or NP. The test result is obtained using
While special attention has been paid to the development of a lateral flow or fluorescence readout within 70 and 40 min,
diagnostic systems based on CRISPR for SARS-CoV-2 nucleic respectively. In addition, examination of NP specimens from
acid detection rapidly, such systems have not addressed COVID-19 patients for further testing of the STOPCovid
mutations and genomic rearrangements related to the virus’s method showed that 12 positive specimens and five negative
nucleic acid. It is well-known that RNA viruses frequently specimens out of three replicates could be detected by
mutate to avoid attacks from hosts’ immune systems. STOPCovid. The STOPCovid method is a valuable technique
Numerous genomes of SARS-CoV-2 have been sequenced, for point-of-care diagnostic systems development for detecting
and different mutations have been identified, indicating that SARS-Cov-2 and has the ability to assist in measures of test−
the coronavirus will constantly adapt to its host. Especially, trace−isolation to end COVID-19 spread and restore public
such mutations have been reported in gene sequences that are health.85 Zhang et al.87 developed a protocol to improve and
focal areas for SARS-CoV-2 diagnostic tests, which can affect advance the diagnosis of COVID-19, using a CRISPR-based
the efficiency of qRT-PCR assays.110−113 Most noticeably, SHERLOCK method. Using artificial RNA fragments of the
mutations in the gRNA binding site can trigger mismatches virus, they were able to identify COVID-19 target sequences in
ACS Sens. 2021, 6, 1430−1445
the 20 and 200 aM range (10−100 copies per microliter of
nucleotide discrimination
ability to single nucleotide
high specificity for single

ability to distinguish one
ability to distinguish one

input). The established protocol can be carried out in less than
nucleotide between
nucleotide between
1 h using RNA extracted from clinical samples, without the
variation detection
accuracy
need for advanced laboratories.87
■ FNCAS9-BASED DIAGNOSIS OF SARS-COV-2
targets
targets
With the aim of rapid and accurate nucleic acid detection,
Azhar et al.114 developed the FnCas9 editor linked uniform
can only be used for RNA targetsneed to convert DNA to RNA to identify DNA
detection assay (FELUDA) procedure, which uses a highly
targetsrestriction of protein activity due to the secondary structure of RNA

accurate enzymatic readout to diagnose nucleic acid sequences.
Since the FELUDA approach does not require complicated
instruments and can be an effective and convenient alternative
perform collateral ssDNA cleavage only in sequences containing PAM

to diagnostic methods such as PCR-based methods, it was
examined for the diagnosis of SARS-CoV-2. It was observed
that the use of FELUDA for the detection of SARS-CoV-2
using the specific FnCas9 RNP complex leads to clear
signatures of the SARS-CoV-2 sequence in synthetic DNA.
Surprisingly, the FELUDA method was able to differentiate
shortcomings
between sequences of SARS-CoV-2 and SARS-CoV-1 which
differed in one nucleotide. Ultimately, after effective detection
of viral signatures from small amounts of total RNA nucleic
acid collected from SARS-CoV-2 cases within 1 h, they verified
target site must be near the PAM
target site must be near the PAM

the validity of such lateral flow tools as a fast, inexpensive, and
machine-independent alternative to the existing detection
procedures.114
■ CRISPR/CAS3-BASED DIAGNOSIS OF SARS-COV-2

Recently, several groups have documented that class I, type I
CRISPR systems from Escherichia coli and Thermobifida f usca,
Table 2. Comparison of the Cas Proteins Properties Used in the Diagnosis of SARS-CoV-2
both using Cas3 enzyme and the crRNA-bound complex, can

trigger targeted cleavage of DNA in human cells by long-range
Cas12a= TTTN
Cas12b = TTN
deletions.80,115,116 Yoshimi et al.80 established an in vitro

flanking site
protospacer
nucleic acid diagnostic tool based on Cas3, Cas3-operated

PAM
(PFS)
nucleic acid detection N (CONAN). The CONAN tool is a

NGG
AAG
sensitive, fast, and device-free diagnostic system for SARS-
CoV-2 detection in combination with isothermal amplification
DNA (dsDNA
RNA (ssRNA)
methods. To assess the efficiency of the CONAN system in

or ssDNA)
molecule
target
detecting nucleic acids of SARS-CoV-2, purified RNA nucleic

acids from NP swabs, including 10 positive COVID-19
DNA
DNA
samples (confirmed by PCR) and 15 negative samples

(confirmed by PCR), were evaluated using CONAN RT-
amino acids
amino acids
amino acids
LAMP and DETCTR RT-LAMP. Tests conducted using the

∼1100−1300
1629 amino
length
900−1300
700−1100
CONAN RT-LAMP system resulted in SARS-Cov-2 detection

acids
in nine out of 10 positive samples as well as the detection of

one sample from negative samples. In general, the SARS-CoV-
2 detection rate by the CONAN RT-LAMP system was found
sp., Lachnospiraceae sp., Prevotella sp.
to be comparable to the DETCTR RT-LAMP system.80 Table

Francisella novicida, Acidaminococcus
flavefaciens, Leptotrichia buccalis

Leptotrichia shahii, Ruminococcus
2 summarizes the characteristics of all Cas proteins used to

diagnose SARS-CoV-2 infection, including Cas12, Cas13,
FnCas9, and Cas3.
organism
■
Francisella novicida
SUPERIORITIES AND SHORTCOMINGS OF

Escherichia coli
CRISPR-BASED DIAGNOSTIC SYSTEMS

Similar to the Cas9 protein, Cas12a detects DNA sequences
and makes double-stranded breaks in the target site. Cas12
endonuclease, especially LbCas12a, causes nonspecific cleavage
type VI
-type V
CRISPR
type II
of ssDNA molecules after binding to its target genomic region,

type I
class II-
class II-
class II
class I-
class
which ultimately leads to degradation of other ssDNAs in the

vicinity. This Cas12 activity led to the development of the
DETECTR method and was used for molecular detection. The
FnCas9
Cas13a
Cas12
Cas3
DETECTR is similar to RT-PCR in terms of accuracy but

surpasses RT-PCR in speed to a final result. In addition, the
ACS Sens. 2021, 6, 1430−1445
collateral cleavage activity of the Cas13 protein also led to the based diagnosis.68,119 Cas13 protein is related to class 2
development of the SHERLOCK method. This method uses CRISPR systems and is capable of recognizing and cutting
the nonspecific activity of the Cas13 protein to cleave the RNA sequences. DNA sequences can be converted to RNA
fluorescent RNA reporter.27,60,66 Both DETECTR and using the T7 promoter to detect DNA sequences using Cas13
SHERLOCK methods can be adapted for detection by lateral nuclease.66 This nuclease does not require a PAM sequence,
flow dipsticks. In general, there are challenges in the diagnosis which makes Cas13 more flexible. However, some of Cas13
of the COVID-19 virus, such as access to staff safety facilities, orthologs require a protospacer flanking site (PFS). There are
collection of samples, extraction of virus genomes, and access four types of Cas13 protein, including Cas13a−d; Cas13a and
to extraction kits and reagents in both RT-PCR and CRISPR- Cas13b are commonly used in engineering and diagnostics.
based diagnostic methods. However, one of the advantages of Like Cas12, the Cas13 protein has collateral activity which is
CRISPR-based diagnostic systems over RT-PCR is the use of used for diagnostic applications such as reporter molecule
isothermal amplification methods that eliminate the need for cutting.66,120,121 Cas9 endonuclease is also member of class II
thermocycling.117 Other advantages are high speed to achieve CRISPR systems and is commonly used in genome editing.
the final result, specificity to target single nucleotides, no need This enzyme specifically identifies and cleaves DNA sequences
for specialized laboratory equipment, and the use of accessible through the guide sequence. Recently, the Francisella novicida-
reporting techniques such as lateral flow strips. The ability of derived Cas9 protein called FnCas9 has been used to detect
the CRISPR systems to quickly recognize a variety of SARS-CoV-2. Since the FnCas9 protein requires a PAM
infectious diseases caused by emerging viruses such as sequence, PAM can be engineered into the primers to target
coronavirus is one of the significant capabilities of this system. and identify non-PAM regions. The FnCas9 enzyme is capable
In addition, the CRISPR system is able to detect coronavirus of cutting DNA sequences within the 10−50 °C temperature
mutant types with high accuracy. In CRISPR-based diag- range. Considering the sensitivity of FnCas9 to mismatch, it
nostics, since protected areas are used to design the gRNA, the can be used to detect single nucleotide variations.114,122 Cas3
diagnostic platform will be able to diagnose the disease even if endonuclease belongs to class I CRISPR systems which are
the virus genome is mutated. Variations in viral load at capable of identifying and cutting DNA sequences. This
different stages of the disease is another challenge; that is, protein has a length of approximately 1100 amino acids and
when the viral load is low, conventional diagnostic tests may requires a PAM sequence to cut the target site.80
not be able to detect the viral infection and may need to
increase the viral load, which results in false negative results.
However, CRISPR-based diagnostic systems rely on the
■ CONCLUSIONS AND PERSPECTIVES
The increasing number of deaths caused by the COVID-19
detection of the viral genome, so they are able to diagnose outbreak has caused major concern worldwide. One of the
the infection at every stage of the disease and do not require confounding facets of COVID-19 is that it presents a wide
additional confirmatory tests.24,109,118 In addition, the multi- range of symptoms from patient to patient. Therefore, highly
plexing ability of the CRISPR system allows it to distinguish sensitive, specific, and precise approaches need to be
between multiple viral pathogens or even different viral established for early detection and thus better management
serotypes in the same sample.65 Off-target occurrence, where of COVID-19. The discovery of the gene-editing toolkit known
nonspecific binding of gRNA to the virus genome leads to as CRISPR has reshaped biotechnology and biotechnology-
misinterpretation of results, is one of the main challenges of based medical diagnostic tests. One of the appealing features of
the CRISPR system. Therefore, in order to reduce the off- CRISPR is that it can be programmed to target almost any
target effects, it is necessary to use specialized tools for gRNA region of interest within the desired genome. Known for its
design to select the best one(s).24
■
genome manipulation applications, the CRISPR technique
possesses a broad range of other applications and has been
OVERVIEW ON CAS PROTEINS USED IN effectively used to diagnose SARS-CoV-2 in recent studies.
SARS-COV-2 DETECTION Speed, accuracy, power, low cost, sensitivity, and versatility are
Special attention has recently been drawn to the CRISPR the main features of this promising tool in the quest to banish
revolution for the early detection of SARS-CoV-2, which is still disease from humans. Recently, CRISPR-based diagnostic
rapidly spreading and has affected millions of people systems have been developed that include the use of Cas12a
worldwide. In general, diagnostic systems based on CRISPR and Cas13 enzymes. Like Cas9, the Cas12a nuclease binds to
consist of two main elements: (1) Cas protein-guide RNA the desired genomic area via a gRNA and makes the break. In
sequence and (2) modified nucleic acids used as reporters. practice, however, the difference between Cas12 and Cas9 is
Reporters generate a visual signal when they cut, which is used that when Cas12 begins to cleave the target DNA, it also starts
in detection. Once the specified nucleic acid sequences are to cleave nonspecifically neighboring single-stranded DNA, so
detected and cut, reporter molecules are subsequently cut and that a cleaved fluorescent reporter around the target genome
generate a visual signal. Cas proteins used by different groups could be detected. Cas13 nuclease has a similar function to
to recognize the nucleic acid of SARS-CoV-2 include Cas3, Cas12a, but unlike Cas12a it acts on RNA sequences. A
FnCas9, Cas12a and Cas12b, and Cas13a. Particularly, most CRISPR/Cas13-based system can be programmed to target
CRISPR-based SARS-CoV-2 diagnostic studies have enjoyed SARS-CoV-2 RNA sequences where the Cas13 nuclease can
Cas12 protein. The Cas12 protein belongs to the class II attack via an RNA guide and cleave the target region to destroy
CRISPR systems which recognize and cleave DNA (dsDNA or viruses. Following the outbreak of COVID-19 disease, different
ssDNA) sequences. This nuclease has high specificity for groups have developed various diagnostic systems to diagnose
dsDNA sequences so that it can distinguish very similar SARS-CoV-2 nucleic acid using the nonselective cutting
dsDNA sequences from each other, while it does not have this activity of Cas12a and Cas13 nucleases. Quickly obtaining
ability for ssDNA sequences. Cas12a and Cas12b are the two results is one of the great advantages of CRISPR-based
subtypes of the Cas12 protein commonly used in CRISPR- diagnostic systems. Another exciting fact of employing
ACS Sens. 2021, 6, 1430−1445
ACS Sensors
■
pubs.acs.org/acssensors Review
CRISPR-based diagnostic systems is that they do not need AUTHOR INFORMATION

sophisticated laboratory equipment, so they can be easily used
even in areas with poor economic conditions. Due to the Corresponding Authors
programmability of the CRISPR system, it can be harnessed in Hossein Danafar − Zanjan Pharmaceutical Biotechnology
many fields. For example, the CRISPR system can be used to Research Center, Zanjan University of Medical Sciences,
identify key factors involved in the pathogenicity of SARS- Zanjan, Iran; Joint Ukraine−Azerbaijan International
CoV-2 in the host cells, which could be a touchstone in Research and Education Center of Nanobiotechnology and
understanding the mechanism of COVID-19 pathogenesis and Functional Nanosystems, Baku, Azerbaijan; orcid.org/
in finding the drug. However, there are challenges and 0000-0001-8956-7895; Email: [email protected]
problems in using the CRISPR/Cas system for nucleic acid Saeed Kaboli − Department of Medical Biotechnology, School
detection. For example, one of the common problems in of Medicine, Zanjan University of Medical Sciences, Zanjan,
CRISPR/Cas mediated detection is the limited number of Iran; Email: [email protected]
detectable sequences. Cas proteins recognize target sequences Hamed Nosrati − Zanjan Pharmaceutical Biotechnology
on the desired nucleic acid through the guide RNA that must Research Center, Zanjan University of Medical Sciences,
complement the target region. On the other hand, depending Zanjan, Iran; Joint Ukraine−Azerbaijan International
on the type of Cas protein used, the presence of a sequence Research and Education Center of Nanobiotechnology and
called PAM is critical to create a functional correct form Functional Nanosystems, Baku, Azerbaijan; orcid.org/
between the guide RNA/Cas complex and the target region. 0000-0002-7487-8188; Email: nosrati.hamed2020@
Another important issue is mismatch tolerance, in which the gmail.com
tolerance of mismatches between the spacer sequence and the Murali M. Yallapu − Department of Immunology and
target sequence depends on the Cas protein family. The Microbiology, School of Medicine and South Texas Center of
number of mismatches as well as their location relative to the Excellence in Cancer Research, School of Medicine, University
PAM sequence are determinants of the overall mismatch of Texas Rio Grande Valley, McAllen, Texas 78504, United
tolerance, so mismatches farther from the PAM sequence are States; orcid.org/0000-0002-0073-8828;
more tolerated. The Cas13 protein family requires PFS and is Email: [email protected]
capable of PAM-free detection. Standardization is also a very João Conde − NOVA Medical School, Faculdade de Ciências
important factor in effective detection with the CRISPR Médicas and Centre for Toxicogenomics and Human Health
system. Protocols need to be standardized to ensure that all (ToxOmics), Genetics, Oncology and Human Toxicology,
operators achieve the same result. Since the level of nucleic NOVA Medical School, Faculdade de Ciências Médicas,
acid concentration in patient samples is associated with disease Universidade Nova de Lisboa, Lisboa, Portugal;
progression, in addition to qualitative diagnosis, quantitative orcid.org/0000-0001-8422-6792; Email: joao.conde@
diagnosis is also very important. CRISPR-based diagnostic nms.unl.pt
platforms such as SHERLOCK, DETECTR, and HOLMES
are only capable of qualitative detection and cannot quantify Authors
the desired nucleic acid content. Many CRISPR-based Hossein Rahimi − Department of Medical Biotechnology,
diagnostic systems still require amplification of the target School of Medicine and Zanjan Pharmaceutical
nucleic acid prior to detection. Conventional methods for Biotechnology Research Center, Zanjan University of Medical
nucleic acid amplification, such as PCR, are not suitable for use Sciences, Zanjan, Iran
in POC systems due to the need for thermocycling as well as Marziyeh Salehiabar − Drug Applied Research Center, Tabriz
the need for several temperature steps leading to prolonged University of Medical Sciences, Tabriz, Iran; Joint
reactions. In these cases, isothermal amplification methods can Ukraine−Azerbaijan International Research and Education
be used in the absence of equipment and skilled operators. Center of Nanobiotechnology and Functional Nanosystems,
LAMP and RPA techniques are the most common isothermal Baku, Azerbaijan
amplification systems used in POC diagnostic systems. The Murat Barsbay − Hacettepe University, Department of
low temperature requirement is one of the advantages of the Chemistry, Ankara 06800, Turkey; orcid.org/0000-0003-
RPA technique over LAMP. Problems such as dimer primer 0788-4446
formation as well as nonspecific replication products are Mohammadreza Ghaffarlou − Hacettepe University,
caused by low temperatures. However, if the CRISPR system Department of Chemistry, Ankara 06800, Turkey
with high specificity for its target area is used, these nonspecific Taras Kavetskyy − Joint Ukraine−Azerbaijan International
and undesirable products will not interfere with the reaction Research and Education Center of Nanobiotechnology and
and ultimately the reaction result. Functional Nanosystems, Baku, Azerbaijan; Department of
The defensive feature of CRISPR can be applied for the Surface Engineering, The John Paul II Catholic University of
design and establishment of effective antiviruses against SARS- Lublin, 20-950 Lublin, Poland; Drohobych Ivan Franko
CoV-2. This approach can be used to create antivirals against State Pedagogical University, 82100 Drohobych, Ukraine;
other possible new viruses that we may encounter in the future, orcid.org/0000-0002-4782-1602
allowing us to respond quickly to pandemics hereafter. Ali Sharafi − Zanjan Pharmaceutical Biotechnology Research
Moreover, using the easy reprogramming capability of the Center, Zanjan University of Medical Sciences, Zanjan, Iran;
CRISPR system, multiple guide RNAs can be used to ensure Joint Ukraine−Azerbaijan International Research and
that the target sequence is identified even if the virus mutates. Education Center of Nanobiotechnology and Functional
In addition to being a hope for coronavirus treatment, CRISPR Nanosystems, Baku, Azerbaijan
is ambitiously in the race for diagnostics and testing and will Soodabeh Davaran − Drug Applied Research Center, Tabriz
likely leave an effective and lasting impact on the current University of Medical Sciences, Tabriz, Iran; Joint
pandemic. Ukraine−Azerbaijan International Research and Education
ACS Sens. 2021, 6, 1430−1445
Center of Nanobiotechnology and Functional Nanosystems, (16) Broughton, J. P.; Deng, X.; Yu, G.; Fasching, C. L.; Servellita,
Baku, Azerbaijan V.; Singh, J.; Miao, X.; Streithorst, J. A.; Granados, A.; Sotomayor-
Subhash C. Chauhan − Department of Immunology and Gonzalez, A.; Zorn, K.; Gopez, A.; Hsu, E.; Gu, W.; Miller, S.; Pan,
Microbiology, School of Medicine and South Texas Center of C.-Y.; Guevara, H.; Wadford, D. A.; Chen, J. S.; Chiu, C. Y. CRISPR−
Excellence in Cancer Research, School of Medicine, University Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38,
of Texas Rio Grande Valley, McAllen, Texas 78504, United 870.
(17) Radmard, S.; Reid, S.; Ciryam, P.; Boubour, A.; Ho, N.; Zucker,
States J.; Sayre, D.; Greendyke, W. G.; Miko, B. A.; Pereira, M. R.; Whittier,
Complete contact information is available at: S.; Green, D. A.; Thakur, K. T. Clinical utilization of the FilmArray
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Ying Hui, L.; Xu, F. Advances in digital polymerase chain reaction
The authors declare no competing financial interest.
■
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■
(21) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.;
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CRISPR Systems for COVID-19 Diagnosis

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T he emergence of severe acute respiratory syndrome

© 2021 American Chemical Society https://dx.doi.org/10.1021/acssensors.0c02312

molecular biology. The beneﬁts of NGS technology including

■ CRISPR/CAS-BASED SYSTEMS FOR COVID-19

ACS Sens. 2021, 6, 1430−1445

the 20 and 200 aM range (10−100 copies per microliter of

high speciﬁcity for single

ability to distinguish one

■ FNCAS9-BASED DIAGNOSIS OF SARS-COV-2

targetsrestriction of protein activity due to the secondary structure of RNA

perform collateral ssDNA cleavage only in sequences containing PAM

target site must be near the PAM

target site must be near the PAM

■ CRISPR/CAS3-BASED DIAGNOSIS OF SARS-COV-2

both using Cas3 enzyme and the crRNA-bound complex, can

deletions.80,115,116 Yoshimi et al.80 established an in vitro

nucleic acid diagnostic tool based on Cas3, Cas3-operated

nucleic acid detection N (CONAN). The CONAN tool is a

methods. To assess the eﬃciency of the CONAN system in

detecting nucleic acids of SARS-CoV-2, puriﬁed RNA nucleic

samples (conﬁrmed by PCR) and 15 negative samples

LAMP and DETCTR RT-LAMP. Tests conducted using the

CONAN RT-LAMP system resulted in SARS-Cov-2 detection

in nine out of 10 positive samples as well as the detection of

to be comparable to the DETCTR RT-LAMP system.80 Table

flavefaciens, Leptotrichia buccalis

2 summarizes the characteristics of all Cas proteins used to

SUPERIORITIES AND SHORTCOMINGS OF

CRISPR-BASED DIAGNOSTIC SYSTEMS

of ssDNA molecules after binding to its target genomic region,

which ultimately leads to degradation of other ssDNAs in the

DETECTR is similar to RT-PCR in terms of accuracy but

CRISPR-based diagnostic systems is that they do not need AUTHOR INFORMATION

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