Molecular Biotechnology (2023) 65:146–161

https://doi.org/10.1007/s12033-021-00431-7

REVIEW

The Trend of CRISPR‑Based Technologies in COVID‑19 Disease: Beyond

Genome Editing
Zeinab Yousefi Najafabadi1,2,3 · Songwe Fanuel4 · Reza Falak3 · Saeed Kaboli5 · Gholam Ali Kardar1,2

Received: 29 September 2021 / Accepted: 22 November 2021 / Published online: 29 January 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and
treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art
technologies in genomic and proteomic engineering. CRISPR–Cas system is one of the emerging techniques in the field of
biotechnology. To date, the CRISPR–Cas system has been extensively applied in gene editing, targeting genomic sequences
for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches.
With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools
such as the CRISPR–Cas system for use in diagnostic emergencies. This system can compete with other existing biotech-
nological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this
article, we review the various platforms of the CRISPR–Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic
procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine
development.

Keywords CRISPR–Cas systems · SARS-CoV-2 · Diagnosis · Anti-viral approach · Genome-wide association study ·
Animal model

Introduction for the third large-scale human outbreak of its family in the
last two decades. This zoonotic virus is a non-segmented
The SARS-CoV-2 virus, a member of the Coronaviri- enveloped positive-sense single-stranded RNA virus with
dae family, Betacoronavirus genus, and the causative agent a large genomic size that has high animal-to-human and
of Coronavirus Disease 2019 (COVID-19), is responsible human-to-human transmission compared to its family
members [1]. The genomic sequence of this new emerging
virus has a high rate of mutation and recombination, due to
* Gholam Ali Kardar its unique self-replication phenomenon [2]. At the begin-
gakardar@tums.ac.ir
ning of the pandemic, the complete sequence of the viral
Zeinab Yousefi Najafabadi genome was shared through metagenomic approaches and
z-yousefin@razi.tums.ac.ir
the three-dimensional structure of its key proteins was deter-
1
Department of Medical Biotechnology, School of Advanced mined [3]. The fourteen open reading frames (ORF) of the
Technologies in Medicine, Tehran University of Medical SARS-CoV-2 genome encode sixteen nonstructural proteins
Sciences, Tehran, Iran to form replicase complex (such as 3-chymotrypsin-like pro-
2
Immunology, Asthma Allergy Research Institute (IAARI), tease (3CLpro), papain-like protease (PLpro), helicase, and
Tehran University of Medical Sciences, Tehran, Iran RNA-dependent RNA polymerase (RdRp), nine accessory
3
Department of Immunology, School of Medicine, Iran proteins, and four structural proteins (spike (S), membrane
University of Medical Sciences, Tehran, Iran (M), envelope (E), and nucleocapsid (N)). Spike protein has
4
Department of Applied Biosciences and Biotechnology, two functional segments (S1 and S2) that are activated by
Faculty of Science and Technology, Midlands State host cell proteases (cathepsin L and transmembrane protease
University (MSU), Gweru, Zimbabwe
serine 2 (TMPRSS2) [4]. In SARS-CoV-2 the receptor-bind-
5
Department of Medical Biotechnology, School of Medicine, ing domain (RBD) of spike protein binds to the human cell
Zanjan University of Medical Sciences, Zanjan, Iran

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Molecular Biotechnology (2023) 65:146–161 147

receptors, Angiotensin-Converting Enzyme 2 (ACE2), and [10]. Types II, V, and VI from class II of the CRISPR/
defines the tropism and the pathogenicity of the virus, While Cas system have enzymatic activity with a similar nucle-
the spike protein of other human coronaviruses can bind to ase domain in their effector protein through the detection
the other cellular entry receptors such as aminopeptidase N of target DNA or RNA sequences. In CRISPR/Cas com-
(APN) and dipeptidyl peptidase 4 (DPP4) too. Mutation in plex, the CRISPR RNA (crRNA) is responsible for target
RBD enhances the binding affinity of the virus to human sequence (RNA or DNA) identification and can depend
ACE2 and consequently higher transmissibility compared on protospacer-associated motif (PAM) or protospacer
to SARS-CoV and MERS-CoV [1, 5]. flanking site (PFS) based on CRISPR types. In addition
The creation of new and fast platforms at three compasses to crRNA and Cas complex, type II and type V-B have
of prevention, diagnosis, and treatment of virus infections additional trans-activating RNA (tracrRNA) which medi-
are remarkable biotechnological approaches in the COVID- ates the binding between them. The single-guide RNA
19 pandemic. Therefore, knowing more about the biology (sgRNA) sequence is chimerically designed with about
and pathogenesis of the virus will help in diagnosis, treat- 20 nucleotides complementary of the target sequence,
ment regimens, and vaccine design. Regions of RdRP gene instead of the crRNA-tracrRNA complex, in the labora-
sequences in the ORF1ab, envelope protein gene (E), and tory to use as a biotechnological tool. In these complexes,
nucleocapsid protein gene (N) are hotspot regions used for the nuclease domains of the Cas protein have cleavage
molecular detection of the SARS-CoV-2 virus due to their activity, resulting in blunt or overhang on-target sequences
conserved sequences [2]. The use of intervention strategies, [11, 12]. Table 1 summarizes the three most applied types
such as targeting the virus cell entry pathway, including of class II CRISPR–Cas systems [13–16].
RBD, as well as the virus replication complex, including
RdRp, are critical targets in the design of anti-viral molecu-
lar therapeutic platforms [6, 7].
Applications of CRISPR–Cas Systems
in COVID‑19 Disease
CRISPR–Cas System
Some aspects of the CRISPR system beyond genome
Clustered Regularly Interspaced Short Palindromic editing have been considered by researchers during this
Repeats (CRISPR) and its associated proteins (Cas) are course of the COVID-19 pandemic. In this review, we
encoded by bacteria and archaea as defensive mechanisms screened research articles from PubMed and LitCovid for
against invasive genetic agents such as viruses and plas- CRISPR–Cas system application in SARS-CoV-2 using
mids through a three-stage process of adaptation, matura- ‘‘COVID-19 and CRISPR’’ as keywords for the period
tion, and interference [8, 9]. This two-component system 2020 and 2021. We returned 290 articles and after delet-
(CRISPR array and Cas protein) is classified into class I ing articles that were unrelated, review, and preprint arti-
and class II, including different types and subtypes based cles, we were left with 86 articles focusing on diagnosis,
on computational sequence and protein analysis of their anti-viral therapeutics, preclinical models, and genome-
effector subunits. The discovery of the CRISPR system wide screening. The percent distribution of the diagnosis
has revolutionized biotechnology approaches because of researches and the other categorized research articles is
its genomic sequence recognition and enzymatic cleavage presented via a pie diagram in Fig. 1.
ability to genetic engineering of the genome and beyond

Table 1  Applied types of class II CRISPR–Cas system

Type Subtypes Nuclease domain PAM/PFS Cleavage activity Applications

II (Cas9) II-A HNH and RuvC domains 3′ G-rich motif dsDNA blunt cleavage activity Diagnostic platforms
II-B Genome-wide screening
II-C Animal model designing
V (Cas12) V-A (Cas12a/Cpf1) RuvC and Nuc domains 5′ T-rich motif dsDNA overhang cleavage Diagnostic platforms
V-B (Cas12b/C2c1) RuvC domain activity and ssDNA collateral
cleavage activity
VI (Cas13) VI-A (Cas13a/C2c2) 2 HEPN domains 3′ none G PFS ssRNA overhang cleavage Diagnostic platforms
VI-D (Cas13d) None activity and ssRNA collateral Anti-viral therapy
cleavage activity

PAM protospacer adjacent motif, PFS protospacer flanking site

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148 Molecular Biotechnology (2023) 65:146–161

Antiviral Due to the structure of the CRISPR–Cas system, design-

Genome-wide
screening
theraputics ing different and innovative CRISPR-based diagnostic tools
5%
20% often considers the diversity in classes and types of Cas
Preclinical nucleases. Cas subunit with their enzymatic RNase activity
models
1%
properties along with crRNA can identify the target DNA or
RNA and make cleavage [23]. The most important classes
of this enzyme, applicable in detection, are class II (types
II/Cas9, V/Cas12a/b, and VI/Cas13), with attomolar sen-
sitivity for detecting RNA or DNA viruses. Usually, using
each Cas protein type for virus genome detection should
combine with amplification methods, especially isother-
mal methods, to improve the target sample sequence. Then,
Diagnosis through the crRNA and Cas complex, the target sequence
74% can be identified more accurately with visual fluorescent-
based readout or lateral flow assay (LFA) in final detection
Fig. 1  Diagram of published research articles related to CRISPR- step [10, 24]. In cases with low viral RNA copy numbers, it
based studies for COVID-19 is difficult to interpret by visual detection. On the other hand,
there is a correlation between LFA signals and Ct values in
Real-Time PCR results. So, the visual results can be semi-
CRISPR‑Based Diagnosis Assays quantified through machine learning tools to enhance the
analytical sensitivity. There have been efforts to use smart-
For most in vitro molecular diagnostic assays of infectious phone applications for detection and quantification of POC
agents, such as viruses, the target nucleic acid must first SARS-CoV-2 CRISPR-DX platforms by imaging [25]. In
be amplified. Thermocycling-based amplifications, namely, fluorescent-based readouts using diode laser [26], capturing
polymerase chain reaction (PCR) and real-time-PCR meth- of image by companion smart-phone application [27] and
ods, are the gold standard techniques. Also, isothermal- 3D printing instrument for helping smart-phones detection
based amplification methods, namely, loop-mediated iso- [28] are used to develop the POC purposes.
thermal amplification (LAMP) and recombinase polymerase For detecting the SARS-CoV-2 virus, different CRISPR-
amplification (RPA), are other available rapid nucleic acid based platforms are using the diverse Cas nuclease enzymes
amplification tests. Parameters such as sensitivity, specific- listed in Table 2. The Cas9 endonuclease protein in combi-
ity, efficiency, accuracy, simplicity, cost, and especially in nation with gRNA targets the dsDNA sequence. One of the
this case, the speed of reaction are significant criteria for advantages of using the Cas9 type is the ability to identify
creating a suitable molecular diagnostic platform for identi- single-nucleotide polymorphisms that are useful for virus
fication of agents [17, 18]. The goal of designing diagnostic genotyping as well as low-frequency gene mutations [29,
platforms is receiving point-of-care (POC) methods, consid- 30]. This type of capability has enabled the Cas9 from
ering detection with the least tools and even outdoors away Campylobacter jejuni NCTC11168 (CjeCas9) to be used to
from the laboratory. Therefore, simultaneous detection of distinguish the SARS-CoV-2 and its D614G (­ Asp614 → Gly)
multiple agents in the shortest time is changing approach variant with single-base resolution in patient samples [31].
from routine methods to novel methods [19, 20]. Amplification-free electrochemical detection methods are
One of the new applications of the CRISPR–Cas system, based on binding affinity between the dCas9 (dead activity-
beyond genome editing, is the diagnosis of infectious and Cas9) and the viral genome on graphene-based FET chips,
non-infectious diseases. Since 2017 CRISPR-based diagno- which also have the potential for SARS-CoV-2 detection
sis (CRISPR-DX) platforms based on nucleic acid detection [32].
have been developed. The design of CRISPR-DX platforms The first diagnostic platform to be designed based on
has been able to consider most of the necessary parameters Cas12 was a DNA endonuclease-targeted CRISPR trans
by incorporating other amplification and detection methods reporter (DETECTR) for the detection of HPV in human
to develop optimal systems with high performance [21]. To patient samples. In this platform, the crRNA-Cas12a com-
date, there is an urgent need to develop new POC detec- plex was used to bind and cleave the amplified virus genome
tion methods in the emerging condition of the COVID-19 sequence. Collateral cleavage of a non-specific short ssDNA
pandemic. In 2020 the CRISPR-based detection systems for sequence, which is a fluorophore quencher (FQ) labeled as
SARS-CoV-2 have been able to receive the US food and a reporter was used to visualize this binding by light emis-
drug administration (FDA) emergency use authorization sion [52]. The DETECTR platform has been redesigned
(EUA) approval [22]. for SARS-CoV-2 detection from the extracted RNA of a

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 Table 2  Notable CRISPR–Cas-based diagnostic platforms for SARS-CoV-2 designed in 2020 and 2021
CRISPR–Cas Technique name Attendant methods Target genes Limit of detection Duration time References
type (LOD)
Amplification Detection method
method

Type II (Cas9) Cas9/D10A with CRISPR array- PER Electrochemical Nucleocapsid (N) 5 nM for synthesized Three steps (CRISPR [33]
nickase activity mediated biosensing SARS-CoV-2 reactant, PER reac-
primer exchange genome in the tant, and detection)
reaction-based human cell lysate within 110 min
biochemical circuit
cascades
Molecular Biotechnology (2023) 65:146–161

Francisella novicidae CRISPR/Cas9- RT-RPA Lateral flow visual Simultaneous dual 100 RNA copies per Two steps at 37 °C [34]
Cas9 mediated triple-line readout genes; envelope (E) reaction (25 μl) within 40 min
lateral flow assay and Orf1ab
(TL-LFA)
Campylobacter Leveraging engi- RT-PCR or free- Polyacrylamide gel Spike (S) One copy, or 1.7 aM unspecified [31]
jejuni NCTC11168 neered tracrRNAs amplification electrophoresis or in the original dilu-
(CjeCas9) and on-target Bioanalyzer tion, of RNA com-
DNAs for parallel pared with 3 × ­108
RNA detection copies, or 0.6 nM
(LEOPARD) in the original
dilution, without
preamplification
Type V (Cas12) Cas12a (Cpf1) DNA endonuclease- RT–LAMP Lateral flow visual Nucleocapsid (N) 10 copies per µl of Three steps within [35]
targeted CRISPR readout and envelope (E) nasopharyngeal 30–40 min
trans reporter swab samples
(DETECTR)
CRISPR-based fluo- RT-RPA Fluorescence-based Nucleocapsid (N) 2 copies per µl of Three steps within [36, 37]
rescent diagnosis detection in and envelope (E) nasal swab samples 50 min
system for COVID- 96-well microtiter
19 (COVID-19 plate and adaptable
CRISPR-FDS) to smartphone-read
chip format
In vitro Specific RT-LAMP Lateral flow and Nucleocapsid (N) 10 copies per µl of One and two steps [38]
CRISPR-based fluorescence-based and envelope (E) Nasopharyngeal at 62 °C within
Assay for Nucleic detection swab samples 60 min
acid detection
(iSCAN)
CRISPR–Cas12a RT-RAA​ Fluorescence-based orf1a, orf1b nucle- 10 copies per µl input Three steps within [39]
naked eye readout detection ocapsid (N) and 60 min
(CRISPR–Cas12a- envelope (E)
NER)

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 Table 2  (continued)
150

CRISPR–Cas Technique name Attendant methods Target genes Limit of detection Duration time References
type (LOD)

13
Amplification Detection method
method

Specific enhancer rRT-PCR Fluorescence-based Orf1ab (O) and 1.6 copies per µl of Three steps within [40]
for PCR-amplified detection nucleocapsid (N) pharyngeal and rRT-PCR time and
Nucleic Acid nasopharyngeal CRISPR detection
(SENA) swabs with 95% time
confidence
Manganese-enhanced RT-RAA​ Fluorescence-based Envelope (E) 5 copies with Mn2+ Three steps within [41]
Cas12a detection detection compared to 10 45 min
(MeCas12a) copies with Mg2+
ENHANCE system RT-LAMP Fluorescence-based Nucleocapsid (N) 3–300 copies per µl Three steps within [42]
assay and lateral 40–60 min
flow assay
One-pot visual RT-LAMP Fluorescence-based Spike (S) 5 copies per µl Three steps within [43]
SARS-CoV-2 assay 45 min
detection sys-
tem named
(opvCRISPR)
All-In-One Dual RT-RPA Fluorescence-based Nucleocapsid (N) 5 copies per µl One-step amplifica- [44]
CRISPR–Cas12a assay tion and detection
(AIOD-CRISPR) at room tempera-
ture (37 °C) within
20 min
Microfluidic Isota- RT-LAMP Fluorescence-based Nucleocapsid (N) 10 copies per µl of Within 30 min [45]
chophoresis (ITP)- assay and envelope (E) Nasopharyngeal
CRISPR-based swab samples
Digital warm-start RT-DAMP Fluorescence-based Nucleocapsid (N) 5 copies/μl RNA in One step in Quant- [46]
CRISPR (DWS- assay the chip tenfold Studio 3D digital
CRISPR) higher sensitivity chip initiating at
than tube-based above 50 °C
bulk assay format
Cas12b (C2c1) CRISPR-assisted RT-RAA​ Fluorescence-based RdRp 10 copies per µl one step at 42 °C [47]
detection (CAS- assay within 60 min
detec)
SHERLOCK Testing RT-LAMP Fluorescence-based Nucleocapsid (N) 100 copies/reaction One-step amplifica- [48]
in One Pot (STOP) assay and lateral tion and detection
flow assay at 60 °C within
45 min
Molecular Biotechnology (2023) 65:146–161
 Table 2  (continued)
CRISPR–Cas Technique name Attendant methods Target genes Limit of detection Duration time References
type (LOD)
Amplification Detection method
method

Type VI (Cas13) Cas13a (C2c2) Specific High-sen- RT–RPA and T7 Fluorescence-based Spike (S), nucleopro- 42 RNA copies per Three steps within [26, 49]
sitivity Enzymatic transcription assay and lateral tein (N) replicase reaction of Naso- 60 min
Reporter unlocking flow assay polyprotein 1ab pharyngeal swab
(SHERLOCK) (Orf1ab)
Combinatorial PCR or RT-RPA Fluorescence- spike (S), nucleopro- 104 copies per µl for Three steps [50, 51]
arrayed reactions based assay using tein (N) replicase synthetic targets
Molecular Biotechnology (2023) 65:146–161

for multiplexed mixes of color- polyprotein 1ab

evaluation of coded amplified (Orf1ab)
nucleic acids sequences
(CARMEN-Cas13)
Streamlined high- RT–RPA and T7 Fluorescence-based ORF1a 10 copies per µl with One step [27]
lighting of infec- transcription assay and lateral 100% specificity
tions to navigate flow assay
epidemics (SHINE)
Cas13 assisted RT-RPA and T7 Lateral flow assay S and Orf1ab 200 copies/reaction Two steps dual-heat [25]
saliva-based transcription integrated with a for the S gene with inactivation (37 °C
& smartphone smartphone appli- a corresponding Ct for 10 min and
integrated testing cation value of 35.4 95 °C for 5 min)
(CASSPIT)
PER primer exchange reaction, LAMP loop-mediated isothermal amplification, rLAMP RNA transcription following LAMP, RAA​ recombinase-aided amplification, RPA recombinase polymer-
ase amplification, DAMP dual-priming isothermal amplifications

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152 Molecular Biotechnology (2023) 65:146–161

Step I: Amplification

Reveres transcription &

isothermal amplification

RPA
LAMP
Nasopharyngeal sample Inactivation Extraction of RAA
collection of SARS-CoV-2 SARS-CoV-2 RNA
Amplified DNA for target gene

T7 transcription
Cas9 detection-based Cas12 detection-based Cas13 detection-based

Biotinylated DNA RNA

DNA

Cas12-crRNA Cas13-crRNA ssRNA reporters

ssDNA reporters
Cas9 and FAM- complex complex
labelled
tracrRNA
complex
Step II: CRISPR detection

Binding to target DNA

and activating Binding to target RNA
collateral cleavage and activating
activity collateral cleavage
activity

Binding to target
amplified biotinylated
DNA Cleaved reporters
Cleaved reporters

lateral flow assay lateral flow assay OR Fluorescence-based assay

Blue light
Step III: Visualization

anti-FAM
antibody Cleaved
Biotin-FAM
reporter Cleaved
Quencher-
Fluorescent
Fluorophore
Positive band emission
Sample flow Control band Anti- Control band Positive band anti- reporter
streptavidin Sample flow
rabbit Antibody streptavidin FAM antibody coated
coated coated coated

Fig. 2  An overview of CRISPR-based diagnostic assays with three extraction of RNA of SARS-CoV-2, pass through three steps and can
types of Cas enzymes; Cas9, Cas12, and Cas13 detections with be visualized through the lateral flow assay or fluorescence-based
names of FELUDA, DETECTR, and SHERLOCK, respectively, after assay

respiratory swab, by lateral flow assay. The collaboration of combination with reverse transcriptase-recombinase poly-
this method with reverse transcriptase-loop-mediated iso- merase amplification (RT-RPA) and T7 transcription to
thermal amplification (RT-LAMP) to convert and amplify increase detection accuracy using a quenched fluorescent
RNA virus to DNA effectively increased the detection effi- ssRNA reporter suitable for visual or lateral flow read-
ciency [35]. The advantages of this method are laboratory out [55]. This platform was successful in the diagnosis of
instrument independence and rapidness, along with accept- the SARS-CoV-2 virus during its pandemic. It is the first
able accuracy and specificity [28, 53]. Other DETECTR-like CRISPR-based platform to be used on clinical samples with
platforms for COVID-19 are listed in Table 2. the US FDA-EUA approval and has acceptable results com-
The pioneer of the CRISPR-DX system is Specific High- pared with other diagnostic methods, such as next-genera-
sensitivity Enzymatic Reporter unlocking (SHERLOCK) tion sequencing (NGS) and RT-PCR (q-PCR) [22, 49, 56].
that uses type VI/Cas13 for detection [54]. This platform Discrimination of the wild and mutated SARS-CoV-2, such
was designed to detect pathogens, including viruses, in as D614G mutation with Cas13a-gRNA and Cas12a-gRNA

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Molecular Biotechnology (2023) 65:146–161 153

a An overview of anti-viral therapy for SARS-CoV-2 with CRISPR-based system

Expressed Cas13-
SARS-CoV-2 infection
crRNA complex

Local delivery to
airway cells
SARS-CoV-2
Library of Cas13- RNA genome or mRNA
AAV
crRNA in AAV library
transfer vector for
SARS-CoV-2
conserved genomic Viral genome Blocking viral genome
regions degradation expression

Airway cell infected by SARS-CoV-2

b An overview of pre-clinical animal model production via the CRISPR-Cas9 system

Human ACE2 template

Human ACE2
mACE2
Microinjection into zygote Expressible human
promoter Manipulated zygote

mACE2 promoter
non-expressible
Exon-2 mouse ACE2 ACE2
Intranasal
inoculation of
CRISPR-Cas9 complex SARS-CoV-2

Severe infectious

Humanized ACE2 mouse model Intranasal

inoculation of
SARS-CoV-2

No infectious

Wild-type mouse with mACE2

c An overview of CRISPR genome-wide screening

Virus resistant population pro-viral genes
count

Sequencing &
data analysis

sgRNA

Library transduction

ACE2 positive Genome-wide CRISPR anti-viral genes

count

cell line targeting CRISPR Knock-out

lentiviral library of cells
selected sgRNAs
Sequencing &
data analysis

sgRNA

CRISPR-based platforms, is valuable for monitoring of platforms for SARS-CoV-2 virus detection are listed in
epidemiological analyses and can improve the ability of Table 2. Figure 2 shows the steps of the available three types
POC tests [57, 58]. Other notable CRISPR–Cas13-based of CRISPR-DX platforms.

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◂Fig. 3  a Cas13-crRNA complex through variable delivery systems off-targets in the DNA sequence of virus-infected host
(such as AAV delivery method) can target virus genes (such as cells [15, 64].The CRISPR–Cas13 system is not depend-
ORF1ab, RdRp, S, and N genes) to degrade viral genome and block
genome expression of virus. b Production of humanized ACE2 mouse
ent on the detection of PAM and the crRNA targets ssRNA
model through the microinjection of CRISPR–Cas9 complex and coding sequences of the virus without interfering with
human ACE2 template sequence in mouse zygote to substitute mouse the human transcriptome, causing sequence degradation,
ACE2 gene and express in the lung, intestine, and brain of a mouse interrupting gene expression, and eventually blocking viral
under its promoter. This humanized model is compared with a wild-
type mouse in SARS-CoV-2 infectious conditions. c The pooled or
function [65]. Therefore, using CRISPR–Cas13 is superior
arrayed CRISPR genome-wide screening is done for analysis of top- to CRISPR–Cas9 as an anti-viral programmable inhibition
ranked gene clusters in the pathway of virus infection in host cells. system.
In this direction, designing of sgRNA library for targeting candidate Prophylactic anti-viral CRISPR in human cells (PAC-
host cell gene to production of knocked out cells and challenging with
SARS-CoV-2 virus are done for determining of anti-viral and pro-
MAN), effectively can identify and degrade the virus
viral genes from sensitive and resistant cells sequence and its mutants in the human lung epithelial
cells. The PAC-MAN system uses the class 2, type VI-D,
CRISPR–Cas13d system derived from Ruminococcus fla-
CRISPR‑Based Therapeutics vefaciens XPD3002. PAC-MAN simultaneously identifies
more than 90% of all coronaviruses using a combination of
Conventional therapies for treating viral diseases are usu- 6 crRNAs by identifying protected regions (such as ORF1ab,
ally based on preventing the virus infections by targeting RdRp, and N genes) as well as detecting virus ssRNA in
biomolecules in the entrance pathway up to proliferation. their replication and transcription phases [66]. The com-
In the emergence of viruses, the first step in anti-viral ther- prehensive set of bioinformatics methods for receiving in
apy is drug repurposing to use existing drugs for the ben- silico optimal crRNA candidates can improve the efficiency
efit of treatment in the shortest possible time. The second of laboratory tests for combating SARS-CoV-2 in the least
approach could be target-based drug designing by focus- time [67]. Besides in silico designing and human cell line
ing on the genomics and proteomics of the desired virus tests, using animal models will improve preclinical studies
[59, 60]. Based on these two approaches, drug candidates with the simulation of respiratory infections in vivo and will
for the treatment of SARS-CoV-2 virus infectious usually help to screen the safety and effectiveness of CRISPR-based
focus on disruption of the enzymatic function of the virus platforms for anti-viral approaches [68]. The advantages of
(especially RNA-dependent RNA polymerase), prevention the CRISPR-based anti-viral systems are high flexibility in
of virus endocytosis, blocking viral proteins (envelope, identifying viral sequences, speed of detection, and direct-
membrane, nucleocapsid, and accessory proteins), helping ness against the virus [65, 66]. The Cas13d expression
innate immunity or suppressing the excessive inflamma- vector could be transmitted via the adeno-associated virus-
tory response, and prevention of SARS-CoV-2 replication packaging system (AAV delivery method) with high tropism
[61]. On the other hand, viruses that have new mutations for respiratory tissues and expressed under the induction of
and cause pandemics usually cannot respond to old drugs expression promoters in specific tissues, especially airway
and vaccines. These limitations in the treatment and pre- cells [65]. The basis of the anti-viral CRISPR-based system
vention of the COVID-19 could be applied to other viruses for SARS-CoV-2 is depicted in Fig. 3a.
of the same family, such as SARS and MERS. Therefore,
under these conditions, there is an urgent need for new CRISPR in preclinical Researches
therapies based on targeting the genomic sequences of the
virus. The disruption of gene function in anti-viral ther- Preclinical researches related to COVID-19 require suit-
apy incorporates biotechnological tools, such as siRNA or able animal model that can be infected with the SARS-
CRISPR–Cas system. One of the well-known applications CoV-2 virus and subsequently access mild-to-severe dis-
of the CRISPR–Cas system is the possibility of identifying ease. As in human cells, the target cells in the selected
the DNA or RNA sequence of the virus to cut them and animal should possess ACE2 as the SARS-CoV-2-binding
destroy their function [62]. receptor to let virus cell entrance. Animals such as ham-
Related studies have suggested CRISPR an acceptable sters, ferrets, African green monkeys, cynomolgus, and
candidate for targeting human pathogens such as HIV, hep- rhesus macaques have similar human ACE2 receptors. For
atitis B virus (HBV), herpesviruses, human papillomavirus extensive researches the best candidate is mice because
(HPV), and JC virus (JCV) [63]. The Cas9 and Cas13 as of reducing costs and increasing the number of animals
endonuclease enzyme subunits in the CRISPR system can but mouse ACE2 has much lower binding affinity for the
identify the DNA and RNA of viruses in infected mam- viral spike protein, compared to its human counterpart.
malian cells. Studies have shown that Cas9 has a lower This low binding affinity will develop a mild disease in
cleavage efficacy for ssRNA and also the ability to create mice models that is not suitable for multi-aspect studies

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 Table 3  Genome-wide CRISPR screens of pathways and processes involved in SARS-CoV-2 infection
Screened pathways & process Screened cell line Top-ranked gene clusters in Highlights Similar small molecule References
pathway
Resistant genes Sensitize genes

Cholesterol biosynthesis pathway A549 ATP6AP1 Loss of resistant genes such as RNA-sequencing of cells treated [84]
(part of the endosomal entry ATP6V1A RAB7A reduces viral entry by with amlodipine small molecule
pathway from the vacuolar NPC1 sequestering the ACE2 receptor shows a similar differential
ATPase proton pump, Retromer, RAB7A inside cell gene expression profile as seen
and Commander complexes) CCDC22 in CRISPR knock-out of genes
PIK3C3 in cholesterol biosynthesis
pathway
Molecular Biotechnology (2023) 65:146–161

Rab-GTPase requirements HAP1 SREBP/SCAP MRPS2 SCAP regulates lipid and choles- Fatostatin molecule as SCAP [83]
Glycosylphosphatidylinositol- Huh-7.5-Cas9 HS2ST1 MRPS5 terol homeostasis by sequester- inhibitors shows anti-viral
anchored biosynthesis EIF4E2 MRPS25 ing SREBPs in the ER in the properties
Cholesterol biosynthesis RAB2A MRPS27 presence of sterols 27-Hydroxycholesterol (27OHC)
RAB10 and 25OHC have SARS-CoV-2
RAB14 anti-viral activity in VeroE6
cells
Replication cycle A549 TMEM41B SARS-CoV-2 require TMEM41B None [85]
for replication cycles in cell
Chromatin remodeling Huh7.5 ARID1A TARDBP Viral RNA-RNA and RNA–pro- PFI-3, which targets the bromodo- [80, 81]
Histone modification VeroE6 DYRK1A tein interactions reveal specific mains of the SWI/SNF proteins
Cellular signaling KDM6A SARS-CoV-2-mediated mito- SMARCA4 and SMARCA2
RNA regulation CTSL chondrial dysfunction during SIS3, which targets the pro-viral
ACE2 infection pro-viral genes and gene SMAD3 identified in the
SMARCA4 pathways, including HMGB1, screen
DYRK1A and the SWI/SNF chroma-
KDM6A tin remodeling complex are
HMGB1 SARS-CoV-2 lineage HMGB1
HIRA is critical for SARS lineage viral
CABIN1 entry with critical role in ACE2
TRIP12 expression
BPTF
PIAS2
Glycosaminoglycan biosynthesis Huh-7.5 hepatoma cells (Huh-7.5- SCAP Absolute requirement for the None [86]
SREBP signaling Cas9) TMEM106B VTT-domain containing protein
Glycosylphosphatidylinositol TMEM41B TMEM41B for infection by
biosynthesis VAC14 SARS-CoV-2
Cholesterol biosynthesis ACE2
HMGCS1
MVK
PMVK
RAB6A
RAB10

13
155
 Table 3  (continued)
156

Screened pathways & process Screened cell line Top-ranked gene clusters in Highlights Similar small molecule References
pathway

13
Resistant genes Sensitize genes

Phosphatidylinositol phosphate Huh7.5.1 hepatoma cells TMEM106B Deletions in TMEM106B caused None [87, 88]
biosynthesis B3GALT6 defects in lysosome traffick-
Cholesterol homeostasis B3GAT3 ing, impaired acidification, and
Heparan sulfate biosynthetic B4GALT7 EXT1 reduced levels of lysosomal
genes EXT2 enzymes but its precise molecu-
EXTL3 lar function remains enigmatic
FAM20B
NDST1
SLC35B2
UGDH
XYLT2
SREBP/SCAP
O-glycan biosynthesis HEK293T C1GalT1 Knocking-out N-glycan biosyn- Kifunensine small molecule [89]
N-glycan biosynthesis MGAT1 thesis on Spike-abrogated viral inhibits N-linked glycosylation
entry to reduce viral entry
Cholesterol biosynthesis pathway A549 ATP6AP1 Loss of resistant genes such as RNA-sequencing of cells treated [84]
(part of the endosomal entry ATP6V1A RAB7A reduces viral entry by with amlodipine small molecule
pathway from the vacuolar NPC1 sequestering the ACE2 receptor shows a similar differential
ATPase proton pump, Retromer, RAB7A inside cell gene expression profile as seen
and Commander complexes) CCDC22 in CRISPR knock-out of genes
PIK3C3 in cholesterol biosynthesis
pathway
Rab-GTPase requirements HAP1 SREBP/SCAP MRPS2 SCAP regulates lipid and choles- Fatostatin molecule as SCAP [83]
Glycosylphosphatidylinositol- Huh-7.5-Cas9 HS2ST1 MRPS5 terol homeostasis by sequester- inhibitors shows anti-viral
anchored biosynthesis EIF4E2 MRPS25 ing SREBPs in the ER in the properties
Cholesterol biosynthesis RAB2A MRPS27 presence of sterols 27-hydroxycholesterol (27OHC)
RAB10 and 25OHC have SARS-CoV-2
RAB14 anti-viral activity in VeroE6
cells
Replication cycle A549 TMEM41B SARS-CoV-2 require TMEM41B None [85]
for replication cycles in cell
Molecular Biotechnology (2023) 65:146–161
 Table 3  (continued)
Screened pathways & process Screened cell line Top-ranked gene clusters in Highlights Similar small molecule References
pathway
Resistant genes Sensitize genes

Chromatin remodeling Huh7.5 ARID1A TARDBP Viral RNA-RNA and RNA–pro- PFI-3, which targets the bromodo- [80, 81]
Histone modification VeroE6 DYRK1A tein interactions reveal specific mains of the SWI/SNF proteins
Cellular signaling KDM6A SARS-CoV-2-mediated mito- SMARCA4 and SMARCA2
RNA regulation CTSL chondrial dysfunction during SIS3, which targets the pro-viral
ACE2 infection pro-viral genes and gene SMAD3 identified in the
SMARCA4 pathways, including HMGB1, screen
DYRK1A and the SWI/SNF chroma-
KDM6A tin remodeling complex are
Molecular Biotechnology (2023) 65:146–161

HMGB1 SARS-CoV-2 lineage HMGB1

HIRA is critical for SARS lineage viral
CABIN1 entry with critical role in ACE2
TRIP12 expression
BPTF
PIAS2
Glycosaminoglycan biosynthesis Huh-7.5 hepatoma cells (Huh-7.5- SCAP Absolute requirement for the None [86]
SREBP signaling Cas9) TMEM106B VTT-domain containing protein
Glycosylphosphatidylinositol TMEM41B TMEM41B for infection by
biosynthesis VAC14 SARS-CoV-2
Cholesterol biosynthesis ACE2
HMGCS1
MVK
PMVK
RAB6A
RAB10
Phosphatidylinositol phosphate Huh7.5.1 hepatoma cells TMEM106B Deletions in TMEM106B caused None [87, 88]
biosynthesis B3GALT6 defects in lysosome traffick-
Cholesterol homeostasis B3GAT3 ing, impaired acidification, and
Heparan sulfate biosynthetic B4GALT7 EXT1 reduced levels of lysosomal
genes EXT2 enzymes but its precise molecu-
EXTL3 lar function remains enigmatic
FAM20B
NDST1
SLC35B2
UGDH
XYLT2
SREBP/SCAP
O-glycan biosynthesis HEK293T C1GalT1 Knocking-out N-glycan biosyn- Kifunensine small molecule [89]
N-glycan biosynthesis MGAT1 thesis on Spike-abrogated viral inhibits N-linked glycosylation
entry to reduce viral entry

13
157
158 Molecular Biotechnology (2023) 65:146–161

[69, 70]. The application of transgenic mice can solve this host cell lines can be done by CRISPR–Cas9 genome-wide
problem by inserting the human ACE2 receptor gene into mutagenesis screening. Recently screening of the Norovi-
the mouse embryo genome in an engineered manner that ruses, Zika virus, West Nile virus, and HIV in host cell lines
will pass on to the next generation. In this case, human- has been performed by CRISPRing [77–79].
ized ACE2-incorporated mice will be more sensitive to The necessity to identify the pathogenesis of the SARS-
SARS-CoV-2 infection via intranasal inoculation. Notably, CoV-2 virus pandemic, new screening studies are extended
the pathological outcomes and subsequent lung damages for revealing of viral entry and pathogenesis of host genes
will be similar to human infection [71]. In addition to the through genome-wide pooled or array CRISPR libraries in
biotechnology-based techniques used to produce transgene the appropriate cell lines. In these studies, the candidate host
mice, CRISPR technology could also insert the target genes are determined by systems biology approaches and
gene into the mouse genome by the knock-in process. For host–viral protein interactome [61]. The targeted sgRNA
COVID-19 preclinical studies, the CRISPR–Cas9 knock-in libraries are designed for candidate host genes to produce
technology can help the expression of the human ACE2 knock-out cell lines. These mutant cells will be challenged
receptor in various mouse tissues. Humanized mice sen- by virus and finally, the sequencing of the target region
sitize to SARS-CoV-2 intranasal inoculation and intragas- of the sgRNAs on mutant cell genome will determine the
tric inoculation [72], which is depicted in Fig. 3b. The causative genes of resistance and sensitivity [80–82]. The
production of humanized mice has facilitated anti-viral process of the CRISPR genome-wide screening of SARS-
therapeutic research, the development of vaccines, study- CoV-2 host cell is depicted in Fig. 3c. After knocking-out
ing virus transmission and pathogenesis, and screening steps the genes that make the virus-resistant cells are pro-
severe symptoms in COVID-19. viral, and the genes that make the virus-sensitive cells are
anti-viral. By propagating resistant knock-out cells and
sequencing data analysis, anti-viral drugs would be des-
Genome‑Wide Screening by CRISPR ignable based on these gene pathways [83]. In Table 3, the
CRISPR-based genome-wide screening of important cellular
Functional studies of genes via conditional mutagenesis in pathways and processes to determine top-ranked resistant
mammalian cells have been considered for decades. Genome and sensitive genes involved in SARS-CoV-2 infection is
engineering tools such as Cre-recombinase, RNAi, and reported. In some cases, the candidate small molecules that
designer nucleases such as ZFN and TALENs have been mimic anti-viral gene pathways or blocking pro-viral gene
used for forwarding genetic screening until 2012. The dis- pathways have been specified. The studies show that due
covery of the CRISPR–Cas system as a novel biotechnologi- to the lack of adequate knowledge about specific genetic
cal method of designer nucleases generation is an alternative pathways involved in cytotoxicity and metabolism associated
technique to overcome the limitations. One of the advan- with drug repurposing in SARS-CoV-2, research in this field
tages of genome-wide CRISPR screening is the designing has begun by genome-wide CRISPR.
of sgRNA libraries to target thousands of genes simultane-
ously for pooled or arrayed screening and directly identify
the candidates of desired phenotype [73, 74]. In addition Conclusion
to genome-wide mutagenesis through CRISPR knock-out
approach, this system is a suitable tool for insertion and After less than two years of the COVID-19 pandemic,
deletion of the target genes. Also, site-directed base editing CRISPR technology has shown some potential in all appli-
and up/down gene regulation via gene-specific transcription cations, from diagnosis to treatment of this disease. All of
factors can be done with the new modifications of CRISPR these capabilities are as a result of CRISPR’s high sensi-
system. CRISPRing has provided an opportunity to under- tivity, flexibility, adaptability, and developable platform. In
stand the biology of mammalian cells related to diseases, addition, based on new SARS-CoV-2 mutations and the need
such as cancers and pathogen infectious, including viruses, for newly designed vaccines, we have to find other treatment
and designing the pharmacological studies through the crea- strategies like CRISPR technology for COVID-19 and other
tion of targeted mutant cell libraries [75, 76]. future viral infections. In addition to all advantages of using
Usually the host genes that coordinate the virus entrance the CRISPR system in the detection of the virus variants,
and pathogenesis processes are unknown. The identification producing a preclinical animal model for drug and vaccine
of principal host genes in the viral infection, regulation, or researches, its application in anti-viral therapeutics based
suppression pathway can provide a better understanding of on eliminating the virus in the infectious cells, and studies
the virus function in the host cell and can help to find novel on genes involved in the disease; this system certainly has
potential therapeutic marks such as antagonist drugs and shortcomings.
design an appropriate vaccine. All of these studies in the

13
Molecular Biotechnology (2023) 65:146–161 159

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writing—original draft preparation, ZY; reviewing and editing of the 16. Zetsche, B., et al. (2015). Cpf1 is a single RNA-guided endonu-
manuscript, GK, RF, and SK; all authors read and agreed to the final clease of a class 2 CRISPR-Cas system. Cell, 163(3), 759–771.
version of the manuscript. 17. Esbin, M. N., Whitney, O. N., Chong, S., Maurer, A., Darzacq,
X., & Tjian, R. (2020). Overcoming the bottleneck to widespread
Funding This research was not funded. testing: A rapid review of nucleic acid testing approaches for
COVID-19 detection. RNA, 26(7), 771–783.
18. Shen, M., et al. (2020). Recent advances and perspectives of
Declarations nucleic acid detection for coronavirus. Journal of Pharmaceuti-
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Conflict of interest The authors declare that they have no conflict of 19. Feng, W., et al. (2020). Molecular diagnosis of COVID-19:
interest. Challenges and research needs. Analytical Chemistry, 92(15),
10196–10209.
20. R. Jalandra et al., Strategies and perspectives to develop SARS-
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