CRISPR-based diagnostic in aquaculture: Application, Potential/Opportunities, and

Limitations
Kailash Bohara1*, Ali Parsaeimehr 2, Sujan Bhattarai1
1 The Aquaculture & Fisheries, Center of Excellence, University of Arkansas at Pine Bluff, Pine Bluff,
Arkansas, 71601 USA
2 Department of Agriculture and Natural Resources, College of Agriculture, Science, and Technology,
Delaware State University, Dover, DE, 19901, USA
* Correspondence: Kailash Bohara boharak@uapb.edu
Abstract
Aquaculture is an expanding industry encompassing both inland and sea-based operations, driven
by the increasing demand for aquaculture products. However, the occurrence of various diseases
in the aquaculture industries leads to substantial economic losses amounting to billions of dollars
annually. Rapid diagnosis of diseases is crucial for the early surveillance and treatment of
aquaculture diseases. Although nucleic acid-based diagnostic methods are currently utilized in
aquaculture, they suffer from prolonged turnaround times and a lack of efficient diagnostic
techniques, which hinders the management and treatment of aquatic diseases. In contrast, CRISPR-
based diagnostic methods have emerged as promising alternatives due to their portability,
sensitivity, rapidity, and cost-effectiveness, as demonstrated in human and animal diagnostics.
Consequently, researchers have dedicated efforts in recent years to develop various assays for
diagnosing aquatic diseases, capitalizing on diverse CRISPR-Cas systems, each with their own
strengths and limitations. This review aims to consolidate and summarize the characteristics,
applicability, and limitations of different CRISPR-based diagnostic methods developed
specifically for aquaculture.
Keywords: Fish disease, Aquaculture, Multiplex CRISPR, Amplification, Cas12a

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 1. Introduction
Aquaculture plays a crucial role in addressing global nutrition and food security challenges,
providing approximately 87.5 million tons of aquatic animals annually (FAO, 2022). This
production has been steadily increasing since 1992. However, the combined effects of climate
change and high-density farming practices have disrupted the delicate balance between hosts,
pathogens, and the environment, leading to disease outbreaks (Maulu et al., 2021; Pulkkinen et al.,
2010). These outbreaks result in significant economic losses within the aquaculture industry.
Additionally, pollution caused by humans has a negative impact on the health status of freshwater
and marine aquaculture species (Bohara et al., 2023; Timilsina et al., 2023). High-density and/
recirculating aquaculture practices can also induce stress in the host organisms, making them more
susceptible to diseases (Pulkkinen et al., 2010). Moreover, many diseases affecting aquaculture
lack effective treatment or vaccination methods (Du et al., 2022). Early and accurate diagnosis of
diseases is crucial for farmers to promptly implement control strategies (Bohara et al., n.d.).
The exponential development in technology has led to rapid diagnosis of pathogens and is being
used in veterinary medicine (Suminda et al., 2022). Aquaculture is slowly adapting these
technological innovations for efficient diagnosis. Although, aquaculture industry is embracing
technological innovations, it still lags behind the agricultural sector (Yue & Shen, 2022). In
contrast, the field of human medicine and diagnostics has witnessed remarkable advancements in
recent years. The COVID-19 (Corona virus disease 19) pandemic, for instance, led to the rapid
development of diagnostic assays and vaccines within record-breaking timeframes (Kumari et al.,
2022; Majid et al., 2021). In comparison, aquaculture has struggled to develop efficient diagnostic
tools for several viral diseases, which often require days to months for diagnosis. However,
knowledge gained from research on human and animal disease diagnostics can be harnessed to
develop efficient diagnostic methods for infectious diseases in aquaculture (D. Li et al., 2022).
Currently, polymerase chain reaction (PCR) and cell culture are considered the standard diagnostic
methods by the World Organization for Animal Health (WOAH) due to their high sensitivity and
specificity (WOAH, 2023). Nevertheless, these methods are time-consuming and costly.
Additionally, environmental DNA/RNA research is exploring their potential as early diagnostic
and surveillance tools, although the degradation of nucleic acids in aquatic environments poses
challenges for their detection (Bohara et al., 2022). Additionally, multiplex-PCR, loop-mediated
isothermal amplification (LAMP), DNA microarray, and serology are also being researched as new
molecular tools to identify aquatic diseases rapidly with high sensitivity (Adams & Thompson,
2011). Efforts are underway to develop rapid diagnostic assays for timely pathogen identification
in aquaculture (Gu et al., 2023; MacAulay et al., 2022; Shyam et al., 2022). One of the recent
developments in diagnostic assays which gained popularity within a few years of identification is
the Clustered regularly interspaced short palindromic repeats (CRISPR) (Ghouneimy et al., 2022;
Gootenberg et al., 2018; Kaminski et al., 2021).
CRISPR technology has found widespread applications in aquaculture, including gene editing to
enhance fillet quality (Kleppe et al., 2022), modulation of immune response (Elaswad et al., 2018;
Gratacap et al., 2019), improvement of fertility (Chu et al., 2023), and promotion of growth (Zhong
et al., 2016). In addition to these uses, CRISPR has emerged as a valuable diagnostic tool in the
field of human and animal diseases (Kaminski et al., 2021). In aquaculture, researchers from
Thailand pioneered the development of a CRISPR-based diagnostic tool for the detection of the
White spot syndrome virus (WSSV) (Chaijarasphong et al., 2019). CRISPR-based diagnostics
typically involve the coupling of CRISPR-associated (Cas) enzymes, such as Cas9, Cas12, or

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Cas13, with the target nucleic acid sequence to activate a reporter signal (Kaminski et al., 2021).
To enhance the efficiency of disease detection, CRISPR-Cas-based diagnostics may be combined
with pre-amplification procedures to increase the amount of nucleic acid available for detection
(Gootenberg et al., 2017). However, in cases where pathogens exhibit high nucleic acid
concentrations, pre-amplification-free diagnostics can be employed (Shinoda et al., 2021).
CRISPR-based diagnostics offer the potential to detect low copy numbers of pathogens with high
specificity, sensitivity, and shorter turnaround times compared to conventional methods (T. Huang
et al., 2023; Qi et al., 2022). Promising evidence show CRISPR-based diagnostic techniques have
the potential to revolutionize disease detection and monitoring in aquaculture in the areas such as
(1) Rapid and Specific Pathogen Detection, (2) Point-of-Care Testing, (3) High-throughput
Screening, (4) Detection of Antimicrobial Resistance, and (5) Customizable Detection Platforms.
However, despite the promising applications of CRISPR-based technology in aquatic disease
diagnosis, there is currently a lack of comprehensive reviews discussing its full potential.
Therefore, in this review, we aim to fill this gap by presenting a comprehensive compilation of
various CRISPR-based diagnostic studies in aquaculture. We provide detailed insights into the
potential of these technologies in diagnosing aquatic animal health conditions, while also offering
an overview of the different Cas systems utilized in aquaculture, along with their associated
opportunities and challenges.
2. Methodology
A systematic search and retrieval were conducted following the PRISMA guidelines. The search
was conducted using google scholar, Web of Science, and PubMed databases using the Advanced
search function. ((crispr) AND (diagnostic)) AND (aquaculture), ((CRISPR) AND (diagnostic))
AND (fish) were used as keywords for the search. Duplicate publications identified were removed.
Further screening was performed by eliminating irrelevant studies. Screened papers were retrieved
for full access.
Literature review results
Since the discovery of CRISPR as a diagnostic tool many researchers have developed assays to
detect the different pathogens affecting humans and animals. The publications related to CRISPR-
mediated diagnostic are constantly increasing each year since 2019. There are more than 300
articles published in 2023 alone related to CRISPR as a diagnostic tool. Aquaculture is also seeing
growth in such studies and development each year. Different Cas enzyme and amplification
methods are being used to develop efficient and sensitive systems. Despite the development in
research, there is still a lack of coordination between different agencies for commercial application.
In addition to that, out of 24 different studies conducted, 16 assays were developed with Cas12
endonuclease, 7 with Cas13, and 1 study with Cas14.
3. Application of CRISPR in aquatic disease diagnostic
CRISPR is one of the forefront biotechnological advancements used in genome editing (Adli,
2018), diagnostics (Chaijarasphong et al., 2019; Kanitchinda et al., 2020), therapeutics (Behr et
al., 2021) and cancer (Khan et al., 2016). After the first use of CRISPR-Cas9 as a genome editing
tool in 2013, its application and research have revolutionized the industry. CRISPR-Cas system is
divided into two different classes namely Class 1 and Class 2. Class 1 CRISPR-Cas system is
mostly found in bacteria and archaea whereas Class 2 system is only present in bacteria (Makarova
& Koonin, 2015). Class 1 CIRSPR-Cas system contains multiple effector molecules whereas Class

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2 contains single effector molecule. Class 2 CRISPR system is more popular among researchers
in gene editing and diagnostics due to its specificity, efficiency, and ease of use. This class is further
divided into three types (Type II, V, and VI) according to their function. Cas9, Cas 12 (Cpf1),
Cas13, and Cas 14 are the Class-2 CRISPR-Cas system widely used in diagnostic and research
due to their unique nature of targets. Cas9 (most widely used for gene editing) targets the double-
strand DNA, Cas13 targets the ssRNA and exhibits collateral cleavage, Cas12 can target both
dsDNA/ssRNA exhibiting collateral cleavage, and Cas 14 targets ssDNA (Makarova & Koonin,
2015).
In 2016, a low-cost CRISPR-based molecular diagnostic tool was introduced to detect the Zika
virus from the plasma of infected monkeys for the first time (Pardee et al., 2016). Initially, dCas9
(dead Cas9) was utilized in earlier diagnostic assays. However, the subsequent discovery of Cas12
and Cas13, which exhibit collateral activity, enabled more efficient and faster detection methods
(J. S. Chen et al., 2018; Gootenberg et al., 2017). In the field of aquaculture, three types of Cas
systems have been utilized to date, namely Cas12, Cas13, and Cas14. A total of 18 studies have
reported on the development of CRISPR-based diagnostic assays in aquaculture, each with distinct
characteristics, as listed in Table 1.
Considerably, the CRISPR-based diagnostic platforms offer a significant degree of customization,
providing researchers with the flexibility to manage the assay designs to suit the specific needs
and challenges of aquaculture disease surveillance and management., and it offers to targeting
different pathogens of interest across a diverse array of aquaculture species, facilitating a nuanced
approach to disease detection and monitoring. In this regard, researchers can design CRISPR
assays to selectively target the genetic signatures of specific pathogens known to afflict aquaculture
species, such as bacteria, viruses, or parasites (F. Huang et al., 2022; C. Li et al., 2021; J. Liu et
al., 2023; Tao et al., 2023). Additionally, the adaptable design of CRISPR-based diagnostics
enables the design of multiplexed assays, which can detect multiple pathogens concurrently in a
single sample. This multiplexing capability not only boosts the effectiveness of disease
surveillance initiatives but also enhances their cost-effectiveness. Consequently, aquaculture
environments can undergo comprehensive screening, enabling the detection of a broad spectrum
of potential threats. Additionally, researchers may employ CRISPR technology to assess
antimicrobial resistance profiles in aquaculture pathogens, guiding the development of evidence-
based strategies for antibiotic stewardship and AMR mitigation. Likewise, CRISPR-based
diagnostics can aid in monitoring the environmental impact of aquaculture operations by detecting
indicators of pollution or ecosystem disruption. In summary, the customizable nature of CRISPR-
based diagnostic platforms holds immense promise for advancing disease surveillance and
management in aquaculture. By enabling tailored assay designs, multiplexed detection
capabilities, and innovative applications in disease control, CRISPR-based diagnostics empower
researchers and aquaculture practitioners to address the complex challenges posed by infectious
diseases in aquatic environments. As research in this field continues to evolve, CRISPR-based
diagnostics are poised to play a central role in promoting the health, sustainability, and resilience
of aquaculture systems worldwide (J. Liu et al., 2023; Sullivan et al., 2019; Tao et al., 2023; Xiao
et al., 2021).

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S Name Preamplif Ass Readout method Diseases LOD Referenc
N ication ay es
steps tim
e
1 RPA- RPA <1 Fluorescence White 200 copies (Chaijara
CRISPR/ h spot sphong et
Cas12a syndrome al., 2019)
virus
(WSSV)
2 CRIPR- NS 20 Fluorescence Red- 100 fm (F.
Cas13 m spotted Huang et
grouper al., 2022)
nervous
necrosis
virus
(RGNNV)
3 CRISPR- RPA 1h Fluorescence Enterocyt 50 copies (Kanitchi
Cas12a ozoon of DNA nda et al.,
fluorescen hepatopen 2020)
ce assay aei (EHP)
4 RPA- RPA 2h Fluorescence/Later Acute 20 (C. Li et
CRISPR/ al flow hepatopan copies/μL al., 2021)
Cas12a creatic for
assay necrosis fluroscence
disease , 200
(AHPND) copies/μL
(0.3 fm/L)
for lateral
flow
5 signal-off PCR 1h Fluorescence Staphyloc 10000 (Tao et
Cas14a1- occus CFU/ml al., 2023)
based aureus
platform
(SOCP)
6 CRISPR- Amplifica 15 Fluorescence Taura 1000000 (J. Liu et
Cas13a tion m syndrome copies al., 2023)
free virus (1.66 pM
7 CRISPR- RPA 1h Fluorescence/Later White 530,000 (Sullivan
based al flow spot copies et al.,
SHERLO syndrome 2019)
CK WSSV
8 RAA- RAA 40 Fluorescence Vibrio 2 copies of (Xiao et
CRISPR/ m vulnificus DNA al., 2021)
Cas12a

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9 RPA- RPA 1h Fluorescence/Later Scale drop 40 copies (Sukonta,
CRISPR/ al flow disease per Senapin,
Cas12a virus reaction Meemett
(SDDV) a, et al.,
2022)
1 RT-RPA- RT-RPA 1h Smartphone/Fluore Tilapia 200 copies (Sukonta,
0 CRISPR- scence/Lateral flow lake virus of RNA Senapin,
Cas12a (TiLV) Taengphu
, et al.,
2022)
1 dualplex dRAA 45 Fluorescence Aeromona 2 copies of (Lin et
1 dRAA- m s DNA al., 2022)
CRISPR/ hydrophil
Cas12a a
1 RPA- RPA <1 Lateral flow Acute 100 copies (Naranitu
2 Cas12a h hepatopan plasmid s et al.,
creatic DNA/100f 2022)
necrosis g genomic
disease DNA
(AHPND)
1 CRISPR- LAMP Fluorescence Vibrio 1.36×102 (L. Wang
3 Cas12a parahaem copies et al.,
olyticus 2022)
1 RPA- RPA 45 Fluorescence Acute 100 copies/ (P. Wang,
4 CRISPR/ m hepatopan reaction Guo, et
Cas12a creatic al., 2023)
necrosis
disease
(AHPND)

1 CRISPR- RPA Fluorescence Enterocyt 10 gene (P. Wang,

5 Cas12a ozoon copies Zhang, et
hepatopen al., 2023)
aei
1 CRISPR- RPA Fluorescence White 10 copies (P. Wang,
6 Cas12a spot per Yang, et
syndrome reaction al., 2023)
virus
(WSSV)
1 LAMP- LAMP 30 Fluorescence Taura 100 copies (Major et
7 CRISPR- m syndrome for WSSV al., 2023)
Cas12b virus and 200
(SHERL (TSV) and copies for
OCKv2) White TSV
spot
syndrome

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 virus
(WSSV)
1 RR-Cas RT-RPA 1.5 Fluorescence/Later Yellow 100 copies (Aiamsa-
8 h al flow/DNAzyme head virus plasmid at et al.,
Colorimetric genotype DNA/ 100 2023)
1 fg RNA
1 CRISPR- RPA 40 Fluorescence/Later Large 5 × 103 (C.
9 Cas12a m al flow yellow copies Zhang et
croaker al., 2024)
iridovirus
2 CRISPR- RPA 45 Lateral flow Vibrio 10 (Y. Wang
0 Cas13a m alginolytic copies/µL et al.,
us 2023)
2 CRISPR- RT-RPA 50 Lateral flow Infectious 9.5 (Rong et
1 Cas12a m hematopoi copies/µL al., 2024)
etic
necrosis
virus
(IHNV)
2 CRISPR- RPA NA Lateral flow Yersinia 10 copies/ (Calderó
2 Cas13a ruckeri µL n et al.,
2024)
2 CRISPR- RPA 25- Fluorescence/Later Grass carp 7.2 × 101 (H. Li et
3 Cas13a 40 al flow reovirus copies/μL al., 2023)
m type 1
(GCRV)
2 CRISPR- RAA NA Fluorescence/Later Largemou 3.1 × 101 (Guang et
4 Cas13a al flow th bass copies/μL al., 2024)
ranavirus

Table 1. Characteristics of different CRISPR-Cas system reported for aquatic pathogen diagnostic.,
fM: femtomolar, pM: picomolar, CFU: colony forming unit (Modified and adapted from Bohara
et al. 2023)
3.1 CRISPR-Cas13 based diagnostic
The Cas13-based diagnostic assay, known as Specific High Sensitivity Enzymatic Reporter
UnLOCKING (SHERLOCK), was introduced by Gootenberg et al. in 2017 and is specifically
designed to recognize RNA targets (Gootenberg et al., 2017). The mechanism of SHERLOCK
involves the rapid polymerase amplification (RPA) or Reverse transcriptase-RPA (RT-RPA) of
double-stranded DNA or RNA, respectively. Subsequently, T7 transcription converts the amplified
DNA into RNA, which is then cleaved by the CRISPR RNA (crRNA)-guided Cas13 enzyme. Due
to its collateral activity, Cas13 also cleaves a probe containing a quencher, resulting in signal
detection (Figure 1). Since its introduction, CRISPR-Cas13 has been employed for the detection
of various diseases, including the Zika virus (Gootenberg et al., 2018), dengue virus (Myhrvold et
al., 2018), bacteria (Shen et al., 2020), parasites (Quansah et al., 2023), and fungi (Morio et al.,

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2020). SHERLOCK, in particular, has gained popularity in the field of diagnostics since its initial
application in 2018.
In the context of aquatic pathogen detection, SHERLOCK was first utilized to detect the White
spot syndrome virus (WSSV) at a sensitivity of up to 2 femtograms of DNA (Sullivan et al., 2019).
Sullivan et al. (2019) also assessed the assay's specificity by testing infected shrimp samples for
other diseases, such as AHPND (Acute hepatopancreatic necrosis disease), IHHNV (Infectious
hypodermal and hematopoietic necrosis virus), EHP (Enterocytozoon hepatopenaei), IMNV
(Infectious myonecrosis virus), and TSV (Taura syndrome virus), all of which yielded negative
results. Additionally, Sullivan et al. (2019) successfully developed a lateral flow strip for field
testing. Another group of researchers from China developed a fluorescent SHERLOCK assay to
detect red grouper necrosis virus (RGNNV), capable of detecting RNA at concentrations as low as
100 femtomolar (fm) (F. Huang et al., 2022). The SHERLOCK system was also employed to detect
the Taura syndrome virus (TSV), a threat to the shrimp industry caused by an RNA virus. This
optimized amplification-free system provided results within 15 minutes at 37°C and demonstrated
a detection limit of up to 106 copies/reaction (J. Liu et al., 2023). Although SHERLOCK can target
both DNA and RNA, its popularity in aquaculture is limited due to its qualitative nature rather than
quantitative (Lou et al., 2022). Nonetheless, SHERLOCK exhibits high potential as a field
deployable test kit for disease detection in remote areas with limited access to laboratory facilities.
Moreover, the CRISPR-Cas13-based diagnostics can also be employed for monitoring
antimicrobial resistance (AMR) in aquaculture pathogens. By targeting specific RNA sequences
associated with antibiotic resistance genes, these assays enable the rapid identification of AMR
strains, guiding antibiotic stewardship efforts and mitigating the spread of resistant pathogens in
aquaculture settings (Singh et al., 2022).

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Figure 1. Working mechanism of various CRISPR-Cas systems used for disease diagnostics.

3.2 CRISPR-Cas12 based diagnostic

The DNA endonuclease-targeted CRISPR trans reporter (DETECTR), also known as CRISPR-
Cas12a (Cpf1), utilizes guide RNA (gRNA) for targeting specific nucleic acid sequences. The
gRNA contains variable regions, also called spacers, which are complementary to the target DNA
sequence, leading to cleavage facilitated by the Cas12a endonuclease (Zetsche et al., 2015). Unlike
Cas9, which cleaves adjacent to the protospacer sequence (PAM) and contains a G-rich 3' end of
the gRNA sequence, Cas12a cleaves five nucleotides upstream of the T-rich 3' end (Makarova &
Koonin, 2015). Similar to Cas13, Cas12a also exhibits collateral cleavage activity, enabling the
cleavage of a quencher-reporter pair and releasing the reporter for detection (J. S. Chen et al.,
2018).
The DETECTR system incorporates pre-amplification stages, such as recombinase polymerase
amplification (RPA) or rolling circle amplification (RAA), to amplify the target sequences before
detection, ensuring accurate and sensitive on-field diagnosis at a constant temperature (37-42°C).
The use of RPA for target sequence pre-amplification offers a time-saving alternative to regular
PCR or qPCR methods (Xiao et al., 2021). The integration of RPA in the diagnostic assay for White
spot syndrome virus (WSSV) significantly increased the detection limit by nearly 5 million-fold
(Chaijarasphong et al., 2019). However, a few researchers have explored pre-amplification-free
CRISPR-Cas systems for disease diagnosis to minimize the need for pre-amplification equipment
(Liang et al., 2021; J. Liu et al., 2023). This method is limited to samples containing a high amount
of the target.
Furthermore, Wang et al. (2023) developed a one-pot CRISPR-Cas12a diagnostic assay by
integrating RPA and CRISPR/Cas12a in a single tube for detecting Acute hepatopancreatic
necrosis disease (AHPND) at a sensitivity of up to 100 copies per reaction. The optimal cleavage
temperature for Cas12a is 37°C, making it compatible with RPA, which can be performed within
the range of 37-42°C (P. Wang, Guo, et al., 2023). In remote conditions, the human body
temperature can also be utilized for pre-amplification and cleavage. The RPA-Cas12a detection
system has been adapted for lateral-flow detection using a FAM-ssDNA-Biotin reporter and FAM-
antibody with gold nanoparticles, resulting in color deposition, which is more feasible than a
fluorescence-based system (Kanitchinda et al., 2020). Another study developed an RT-RPA-
Cas12a system for rapid detection of tilapia lake virus (TiLV) using lateral flow and smartphone
fluorescence. This method can detect as low as 200 copies of RNA within an hour at temperatures
ranging from 37-42°C (Sukonta, Senapin, Taengphu, et al., 2022).
In the field of aquaculture, researchers have extensively utilized the Cas12a-based system for
disease diagnostics. However, detecting targets with low copy numbers using this system can be
challenging. Additionally, Cas12a specifically targets double-stranded DNA, so reverse
transcription is required for RNA-based targets, which presents an additional limitation.
3.3 Other CRISPR-based diagnostics
Cas14a (Cas12e) is a small-size protein (61.52 KDa), which was identified by the Doudna lab.
Despite its small size compared to Cas9 and Cas12, making it potentially advantageous for certain
applications due its efficiency cleavage activity targeting the single-stranded DNA (ssDNA)

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without the requirement of a protospacer adjacent motif (PAM) sequence (Harrington et al., 2018).
This mini-Cas enzyme offers an advantage over the aforementioned systems due to its PAM-
independent activity and its smaller size, facilitating easier delivery (Ge et al., 2021). In the field
of aquaculture, this system has been employed for the detection of methicillin-resistant
Staphylococcus aureus (MRSA) by targeting single-stranded DNA (Tao et al., 2023).
Researchers worldwide are actively developing various CRISPR-based diagnostic assays.
NASBACC (nucleic acid sequence-based amplification (NASBA)-CRISPR cleavage) employs
Cas9 (Pardee et al., 2016), HOLMES (one-hour low-cost multipurpose highly efficient system)
utilizes PCR for target pre-amplification (S.-Y. Li et al., 2018), HOLMESv2 incorporates loop-
mediated isothermal amplification (LAMP) for pre-amplification (L. Li et al., 2019), iSCAN
employs Cas12b endonuclease (Aman et al., 2022), and FLASH (Finding Low Abundance
Sequences by Hybridization) utilizes Cas9 enzyme and PCR as pre-amplification (Quan et al.,
2019). However, there have been no published studies in the field of aquaculture employing these
systems thus far.
4. Potential of multiplex CRISPR diagnostic in aquaculture
Simultaneous detection of multiple targets in a single reaction can provide significant advantages
for diagnosing diseases in aquaculture. Co-infection, a common phenomenon in fish and shellfish
diseases (Kotob et al., 2016), can be better understood by employing a single method for detecting
multiple pathogens. The conventional approach of using multiplex-PCR for diagnostic assays has
been limited by its low amplification efficiency (Elnifro et al., 2000). However, the development
of CRISPR-based multiplexed diagnostic tools has addressed this limitation.
One such tool is SHERLOCKv2, which utilizes different reporters and fluorescence signals to
detect multiple targets in a single reaction (Gootenberg et al., 2018). Despite its advantages,
SHERLOCKv2 has its limitations. To overcome them, the Combinatorial Arrayed Reactions for
Multiplexed Evaluation of Nucleic acids (CARMEN) Cas13-based assay was developed (Figure
2.). This assay enables the detection of over 4000 targets using color-coded droplets in a small
reaction. However, CARMEN requires pre-amplification steps and skilled personnel for
fluorescent microscopy (Ackerman et al., 2020). Although there are challenges, such as the lack
of readily available sequences for many aquatic pathogens, the fast and cost-effective detection
provided by CARMEN makes it promising for aquaculture.
Nevertheless, the implementation of these advanced CRISPR diagnostic technologies in aquatic
pathogen research requires significant investment, as laboratories and scientists in the field of
aquaculture are limited. However, with modifications and optimization, the CRISPR diagnostic
technologies developed for human and other animal research can be adapted for better applicability
in aquatic pathogen detection.

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Figure 2. CARMAN assay working principle to detect multiple targets at a time. A) amplified
targets are pooled, color-coded, and emulsified B-C) Subsequently loaded in a microwell chip D-
F) pairs are identified using fluorescent microscopy to detect the targets.

5. Point of care testing (POCT) potential of CRISPR diagnostics

POCT is crucial for disease diagnosis in aquaculture due to lack of infrastructure and instruments
in rural part of the world. POCT devices can be used in any form of disease diagnosis without the
requirement of sophisticated laboratory or skilled personnel. An ideal POCT device should be
affordable, specific, rapid, simple and with high sensitivity according to World Health
Organization (WOAH, n.d.). WOAH has registered several POCT kits for fish and shellfish disease
diagnosis (https://www.woah.org/en/what-we-offer/veterinary-products/diagnostic-kits/the-
register-of-diagnostic-kits/). These kits are mostly based on PCR and western blot which requires
laboratory condition as well as skilled personnel. However, one lateral flow
immunochromatographic assay developed by Innocreate Bioscience Co., Ltd, Taiwan
(https://www.innocreatebio.com/) has been approved by WOAH for WSSV diagnostic testing with
almost 100% accuracy (H. Huang et al., 2019). These test kits are only developed for certain
diseases in economically important species making it difficult for the farmers growing other
aquatic species. PCR is considered the best method for identification with high specificity and
sensitivity. However, the major hurdle in making PCR a POCT in aquaculture is its temperature
requirement. CRISPR based diagnostic requires single temperature regime and can be integrated
in lateral flow strips as POCT devices. The readout can be observed using colorimetric method in
lateral flow strips containing test line and control line to check the presence or absence of
pathogens (Bai et al., 2019; S. Liu et al., 2023; W. S. Zhang et al., 2021). Additionally, fluorescence
signal sensing can be utilized to check for positive and negative for targeted pathogens in a dark

11

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room using fluorophore and quencher reporting signal in the CRISPR assay (Chaijarasphong et
al., 2019; Major et al., 2023). Electrical signal sensing is not yet used in aquatic pathogen diagnosis
however, CRISPR/Cas13 has been used to detect the signals for SARS-Cov-2 after coupling the
assay with graphene field-effect transistor with high sensitivity up to 0.15 copies/µl (Sun et al.,
2023). Integration of CRISPR based diagnostic assays into portable devices is an effective strategy
for POCT. Novel form of integration strategies are being researched in animal disease diagnosis
including the diagnostic chips, smartphone based detection and DNA assisted diagnosis (Aiamsa-
at et al., 2023; H. Chen et al., 2023; Manessis et al., 2022). Because of their affordability, ease of
use, and capacity to generate visual readout signals without the need for specialist equipment,
CRISPR-based molecular diagnostics have become a viable method for POCT (Gootenberg et al.,
2017). Though improvements are addressing these restrictions, challenges still exist regarding
sample processing, cold-chain storage requirements, assay complexity for unskilled users, and
trouble interpreting signals (Agrawal et al., 2021; Ghouneimy et al., 2022). Detection limits
equivalent to PCR have been obtained by combining CRISPR enzymes such as Cas12 and Cas13
with isothermal amplification techniques (Mahas et al., 2022). Further optimizations like
amplification-free protocols (Fozouni et al., 2021) and integration with microfluidics or mobile
phone-based signal acquisition (Aman et al., 2022) could enable fully integrated sample-in to
digital readout devices. If ongoing efforts to improve usability and accuracy are successful,
CRISPR-based diagnostics have immense potential to expand access to affordable, sensitive
nucleic acid testing globally (Gootenberg et al., 2017).
6. Comparison between CRISPR-Cas based diagnostic and current diagnostic in
aquaculture
PCR and cell culture are widely regarded as the most reliable diagnostic methods in aquaculture
due to their reliability and specificity. However, the process of virus isolation from cells can be
time-consuming, taking anywhere from a few days to months, with confirmation typically
performed using PCR. This delay in obtaining results can lead to significant losses for farmers.
The World Organization for Animal Health (WOAH), formerly known as OIE, plays a crucial role
in establishing rules and regulations for animal health diagnostics (WOAH, 2023).
Bacterial, parasitic, and fungal diseases in aquaculture are diagnosed using biochemical tests and
visual observations, but PCR is considered the most reliable method for confirmatory analysis.
PCR offers high sensitivity by amplifying the target region to detect the pathogen. However, it has
limitations, including the requirement for qualified personnel, sophisticated equipment, optimized
protocols, and the use of time-consuming and costly reagents (Yang & Rothman, 2004). The
emergence of CRISPR-Cas-based disease diagnostic research presents a groundbreaking
opportunity for aquatic animal health professionals and farmers, as it has the potential to
revolutionize the speed and efficiency of disease diagnosis in aquaculture. Systems like
SHERLOCK and DETECTR can provide rapid and cost-effective test results with specific target
detection. These systems utilize specific guide RNA molecules that can precisely target the nucleic
acid from the pathogen with the help of the appropriate Cas enzyme (S.-Y. Li et al., 2018).
In the case of certain viral diseases such as WSSV, nested PCR is used, which further extends the
PCR time (WOAH, 2023). However, the CRISPR-Cas-12a system developed by Chaijarasphong
et al. (2019) and CRISPR-Cas13a developed by Sullivan et al. (2019) can detect the same virus
within an hour using a lateral flow strip (Chaijarasphong et al., 2019; Sullivan et al., 2019). This
technology has the potential to overcome the challenges associated with nested PCR for various

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viruses in the future (Chaijarasphong et al., 2019; Sullivan et al., 2019). Although CRISPR-Cas-
based diagnostics are still in the development phase in aquaculture, large-scale testing and
comparative studies with PCR methods are needed to establish a reliable diagnostic method.
7. Efficient disease management using CRISPR technology.
Efficient disease management through CRISPR technology offers numerous advantages,
particularly in the field of aquaculture. By leveraging CRISPR, aquaculture species can be
genetically engineered to possess enhanced resistance to diseases. By targeting specific genes
associated with susceptibility to pathogens, CRISPR can introduce beneficial genetic traits that
confer resistance or tolerance to diseases, thereby reducing the need for chemical pesticides or
antibiotics. This promotes sustainability and environmentally friendly practices within
aquaculture. Furthermore, CRISPR-based diagnostic assays enable rapid and precise detection of
pathogens, facilitating early diagnosis and intervention (Aiamsa-at et al., 2023; Major et al., 2023;
P. Wang, Guo, et al., 2023; P. Wang, Yang, et al., 2023; P. Wang, Zhang, et al., 2023).
These assays can pinpoint unique nucleic acid sequences of pathogens, allowing for sensitive and
specific detection even at low concentrations. In aquaculture, CRISPR diagnostics play a crucial
role in monitoring the presence of pathogens within aquaculture facilities, enabling proactive
disease management strategies (C. Li et al., 2021; J. Liu et al., 2023; Tao et al., 2023). Additionally,
in vector-borne diseases such as malaria or Zika virus, CRISPR can be utilized to control disease
vectors such as mosquitoes or ticks. Through genetic modification, these vectors can be altered to
reduce their ability to transmit pathogens. CRISPR-based gene drives offer a means to propagate
desired genetic modifications throughout wild populations of disease vectors, potentially leading
to the suppression or elimination of disease transmission (Gootenberg et al., 2017; Pardee et al.,
2016).
Moreover, CRISPR-based approaches combat antimicrobial resistance by targeting and
deactivating resistance genes within bacterial pathogens. This selective cleavage and degradation
of antibiotic resistance genes by CRISPR-Cas systems restore antibiotic effectiveness, curbing the
spread of resistant strains. Furthermore, CRISPR diagnostics swiftly identify antimicrobial-
resistant pathogens, guiding appropriate treatment strategies. In aquaculture, CRISPR serves
biocontrol purposes by engineering beneficial microorganisms that outcompete or antagonize plant
pathogens. By enhancing natural antagonist activity or introducing novel biocontrol agents,
CRISPR aids in the suppression of disease outbreaks and improves aquaculture health system
(Hossain et al., 2022; Parsaeimehr et al., 2022; Singh et al., 2022; Q. Wang et al., 2022). Overall,
CRISPR technology provides versatile tools for efficient disease management across diverse
applications. By harnessing the precision and adaptability of CRISPR-based approaches,
researchers and practitioners can devise innovative strategies to combat infectious diseases, bolster
food security, and safeguard public health.
8. Conclusive remarks and future directions
In conclusion, the development of a diagnostic assay that possesses characteristics such as speed,
portability, reliability, sensitivity, specificity, cost-effectiveness, and independence from
sophisticated equipment remains an opportunity for researchers in the field. The CRISPR-Cas-
based diagnostic system, if made commercially available, holds promise in meeting many of these
desirable characteristics. Particularly noteworthy are the rapid detection time and portability
offered by these assays, enabling prompt implementation of mitigation strategies against diseases

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in aquaculture and potentially saving significant financial losses on a global scale. However, it is
important to acknowledge the limitations of each CRISPR-based diagnostic assay. SHERLOCK,
for instance, requires expertise in reaction component preparation and may necessitate pre-
amplification steps at specific temperatures that may not be readily available in on-farm diagnostic
settings. Additionally, the lack of commercially available aquaculture-specific crRNA, probes, and
oligonucleotides poses a challenge. DETECTR, on the other hand, demonstrates advantages in
terms of accuracy and detection speed, but the high mutation rates of viruses can result in false
negatives with these CRISPR systems. Furthermore, CRISPR-based diagnostics can only identify
known pathogens with available genome data, making it challenging to identify new viruses or
strains without resorting to cell culture and sequencing methods. Despite these limitations,
CRISPR-Cas-based diagnostics have significantly influenced the field of molecular biology and
hold great potential for application in aquaculture. While further research is necessary for their
commercial implementation, the advantages offered by these technologies over conventional
methods are substantial. As such, the ongoing development of CRISPR-Cas-based diagnostics
presents an exciting avenue for enhancing disease diagnosis in aquaculture and advancing the field
as a whole. The future research should focus on:
• OIE and Fish health societies worldwide need to take a closer look at these assays
to optimize the performance and potential of CRISPR based diagnostic assays for
OIE listed pathogens in aquaculture.
• Fostering Collaboration with industry partners to help in commercialization of the
assays in portable form can facilitate the development of user-friendly and efficient
tools for on-farm testing.
• The current reliance on conventional nucleic acid extraction methods, particularly
column extraction, presents a hurdle for on-farm testing. Research should focus on
streamlining this process, possibly through the implementation of one-step
extraction methods that allow lysing pathogens in a single tube.
• The temperature requirements for Cas activation pose another obstacle.
Collaborative efforts with industry stakeholders can aid in the creation of portable
heating blocks, offering a solution to this bottleneck and enhancing the feasibility
of on-site testing in varying environmental conditions.
• Current diagnostic tests developed by researchers mostly focus on pathogens
affecting economically important fish and shellfish species. However, more tests
and research should be focused on OIE-listed pathogens, which can cause high
economic losses in epidemic conditions.
• Cost-effectiveness is a pivotal factor influencing farmers' choices when selecting
diagnostic tests. Globally, many farmers express a preference for tests that are both
affordable and efficient. Future research should prioritize conducting benefit-cost
analyses to provide valuable insights and support for farmers, particularly those in
developing countries. This approach will help ensure that diagnostic tests not only
meet high standards of effectiveness but are also economically accessible, catering
to the financial considerations of farmers worldwide.

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References
Ackerman, C. M., Myhrvold, C., Thakku, S. G., Freije, C. A., Metsky, H. C., Yang, D. K., Ye, S. H., Boehm, C.

K., Kosoko-Thoroddsen, T.-S. F., Kehe, J., Nguyen, T. G., Carter, A., Kulesa, A., Barnes, J. R., Dugan,

V. G., Hung, D. T., Blainey, P. C., & Sabeti, P. C. (2020). Massively multiplexed nucleic acid

detection with Cas13. Nature, 582(7811), Article 7811. https://doi.org/10.1038/s41586-020-

2279-8

Adams, A., & Thompson, K. D. (2011). Development of diagnostics for aquaculture: Challenges and

opportunities. Aquaculture Research, 42(s1), 93–102. https://doi.org/10.1111/j.1365-

2109.2010.02663.x

Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Nature Communications, 9(1),

Article 1. https://doi.org/10.1038/s41467-018-04252-2

Agrawal, S., Fanton, A., Chandrasekaran, S. S., Charrez, B., Escajeda, A. M., Son, S., Mcintosh, R., Bhuiya,

A., de León Derby, M. D., Switz, N. A., Armstrong, M., Harris, A. R., Prywes, N., Lukarska, M.,

Biering, S. B., Smock, D. C. J., Mok, A., Knott, G. J., Dang, Q., … Hsu, P. D. (2021). Rapid, point-of-

care molecular diagnostics with Cas13. medRxiv, 2020.12.14.20247874.

https://doi.org/10.1101/2020.12.14.20247874

Aiamsa-at, P., Nonthakaew, N., Phiwsaiya, K., Senapin, S., & Chaijarasphong, T. (2023). CRISPR-based,

genotype-specific detection of yellow head virus genotype 1 with fluorescent, lateral flow and

DNAzyme-assisted colorimetric readouts. Aquaculture, 574, 739696.

https://doi.org/10.1016/j.aquaculture.2023.739696

Aman, R., Marsic, T., Sivakrishna Rao, G., Mahas, A., Ali, Z., Alsanea, M., Al-Qahtani, A., Alhamlan, F., &

Mahfouz, M. (2022). iSCAN-V2: A One-Pot RT-RPA–CRISPR/Cas12b Assay for Point-of-Care SARS-

CoV-2 Detection. Frontiers in Bioengineering and Biotechnology, 9.

https://www.frontiersin.org/articles/10.3389/fbioe.2021.800104

15

Electronic copy available at: https://ssrn.com/abstract=4815342

Bai, J., Lin, H., Li, H., Zhou, Y., Liu, J., Zhong, G., Wu, L., Jiang, W., Du, H., Yang, J., Xie, Q., & Huang, L.

(2019). Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever. Frontiers

in Microbiology, 10. https://www.frontiersin.org/articles/10.3389/fmicb.2019.02830

Behr, M., Zhou, J., Xu, B., & Zhang, H. (2021). In vivo delivery of CRISPR-Cas9 therapeutics: Progress and

challenges. Acta Pharmaceutica Sinica B, 11(8), 2150–2171.

https://doi.org/10.1016/j.apsb.2021.05.020

Bohara, K., Joshi, P., Acharya, K. P., & Ramena, G. (n.d.). Emerging technologies revolutionising disease

diagnosis and monitoring in aquatic animal health. Reviews in Aquaculture, n/a(n/a).

https://doi.org/10.1111/raq.12870

Bohara, K., Timilsina, A., Adhikari, K., Kafle, A., Basyal, S., Joshi, P., & Yadav, A. K. (2023). A mini review on

6PPD quinone: A new threat to aquaculture and fisheries. Environmental Pollution (Barking,

Essex: 1987), 340(Pt 2), 122828. https://doi.org/10.1016/j.envpol.2023.122828

Bohara, K., Yadav, A. K., & Joshi, P. (2022). Detection of Fish Pathogens in Freshwater Aquaculture Using

eDNA Methods. Diversity, 14(12), Article 12. https://doi.org/10.3390/d14121015

Calderón, I. L., Barros, M. J., Fernández-Navarro, N., & Acuña, L. G. (2024). Detection of Nucleic Acids of

the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-

Based Assay. Microorganisms, 12(2), Article 2.

https://doi.org/10.3390/microorganisms12020283

Chaijarasphong, T., Thammachai, T., Itsathitphaisarn, O., Sritunyalucksana, K., & Suebsing, R. (2019).

Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid

amplification for detection of white spot syndrome virus in shrimp. Aquaculture, 512, 734340.

https://doi.org/10.1016/j.aquaculture.2019.734340

16

Electronic copy available at: https://ssrn.com/abstract=4815342

Chen, H., Zhou, X., Wang, M., & Ren, L. (2023). Towards Point of Care CRISPR-Based Diagnostics: From

Method to Device. Journal of Functional Biomaterials, 14(2), 97.

https://doi.org/10.3390/jfb14020097

Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-

Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (New

York, N.Y.), 360(6387), 436–439. https://doi.org/10.1126/science.aar6245

Chu, W.-K., Huang, S.-C., Chang, C.-F., Wu, J.-L., & Gong, H.-Y. (2023). Infertility control of transgenic

fluorescent zebrafish with targeted mutagenesis of the dnd1 gene by CRISPR/Cas9 genome

editing. Frontiers in Genetics, 14.

https://www.frontiersin.org/articles/10.3389/fgene.2023.1029200

Du, Y., Hu, X., Miao, L., & Chen, J. (2022). Current status and development prospects of aquatic vaccines.

Frontiers in Immunology, 13. https://www.frontiersin.org/articles/10.3389/fimmu.2022.1040336

Elaswad, A., Khalil, K., Cline, D., Page-McCaw, P., Chen, W., Michel, M., Cone, R., & Dunham, R. (2018).

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for

Gene Editing. Journal of Visualized Experiments: JoVE, 131, 56275.

https://doi.org/10.3791/56275

Elnifro, E. M., Ashshi, A. M., Cooper, R. J., & Klapper, P. E. (2000). Multiplex PCR: Optimization and

application in diagnostic virology. Clinical Microbiology Reviews, 13(4), 559–570.

https://doi.org/10.1128/CMR.13.4.559

FAO. (2022). The State of World Fisheries and Aquaculture 2022: Towards Blue Transformation. FAO.

https://doi.org/10.4060/cc0461en

Fozouni, P., Son, S., Derby, M. D. de L., Knott, G. J., Gray, C. N., D’Ambrosio, M. V., Zhao, C., Switz, N. A.,

Kumar, G. R., Stephens, S. I., Boehm, D., Tsou, C.-L., Shu, J., Bhuiya, A., Armstrong, M., Harris, A.

R., Chen, P.-Y., Osterloh, J. M., Meyer-Franke, A., … Ott, M. (2021). Amplification-free detection of

17

Electronic copy available at: https://ssrn.com/abstract=4815342

 SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell, 184(2), 323-333.e9.

https://doi.org/10.1016/j.cell.2020.12.001

Ge, X., Meng, T., Tan, X., Wei, Y., Tao, Z., Yang, Z., Song, F., Wang, P., & Wan, Y. (2021). Cas14a1-mediated

nucleic acid detectifon platform for pathogens. Biosensors and Bioelectronics, 189, 113350.

https://doi.org/10.1016/j.bios.2021.113350

Ghouneimy, A., Mahas, A., Marsic, T., Aman, R., & Mahfouz, M. (2022). CRISPR-Based Diagnostics:

Challenges and Potential Solutions toward Point-of-Care Applications. ACS Synthetic Biology,

12(1), 1–16. https://doi.org/10.1021/acssynbio.2c00496

Gootenberg, J. S., Abudayyeh, O. O., Kellner, M. J., Joung, J., Collins, J. J., & Zhang, F. (2018). Multiplexed

and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science (New York,

N.Y.), 360(6387), 439–444. https://doi.org/10.1126/science.aaq0179

Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., Verdine, V., Donghia,

N., Daringer, N. M., Freije, C. A., Myhrvold, C., Bhattacharyya, R. P., Livny, J., Regev, A., Koonin, E.

V., Hung, D. T., Sabeti, P. C., Collins, J. J., & Zhang, F. (2017). Nucleic acid detection with CRISPR-

Cas13a/C2c2. Science, 356(6336), 438–442. https://doi.org/10.1126/science.aam9321

Gratacap, R. L., Wargelius, A., Edvardsen, R. B., & Houston, R. D. (2019). Potential of Genome Editing to

Improve Aquaculture Breeding and Production. Trends in Genetics, 35(9), 672–684.

https://doi.org/10.1016/j.tig.2019.06.006

Gu, B., Zhuo, C., Xu, X., & El Bissati, K. (2023). Editorial: Molecular diagnostics for infectious diseases:

Novel approaches, clinical applications and future challenges. Frontiers in Microbiology, 14,

1153827. https://doi.org/10.3389/fmicb.2023.1153827

Guang, M., Zhang, Q., Chen, R., Li, H., Xu, M., Wu, X., Yang, R., Wei, H., Ren, L., Lei, L., & Zhang, F. (2024).

Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a. Fish & Shellfish

Immunology, 148, 109517. https://doi.org/10.1016/j.fsi.2024.109517

18

Electronic copy available at: https://ssrn.com/abstract=4815342

Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., Cofsky, J. C., Kyrpides, N. C.,

Banfield, J. F., & Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14

enzymes. Science, 362(6416), 839–842. https://doi.org/10.1126/science.aav4294

Hossain, A., Habibullah-Al-Mamun, Md., Nagano, I., Masunaga, S., Kitazawa, D., & Matsuda, H. (2022).

Antibiotics, antibiotic-resistant bacteria, and resistance genes in aquaculture: Risks, current

concern, and future thinking. Environmental Science and Pollution Research, 29(8), 11054–

11075. https://doi.org/10.1007/s11356-021-17825-4

Huang, F., Shan, J., Liang, K., Yang, M., Zhou, X., Duan, X., Jia, X., Zhao, H., Qin, Q., & Wang, Q. (2022). A

new method to detect red spotted grouper neuro necrosis virus (RGNNV) based on

CRISPR/Cas13a. Aquaculture, 555, 738217. https://doi.org/10.1016/j.aquaculture.2022.738217

Huang, H., Deng, J., Lan, Y., Yang, A., Zhang, L., Wen, S., Zhang, H., Zhang, Y., & Deng, Y. (2019). Detection

of Helminthosporium Leaf Blotch Disease Based on UAV Imagery. Applied Sciences, 9(3), Article

3. https://doi.org/10.3390/app9030558

Huang, T., Zhang, R., & Li, J. (2023). CRISPR-Cas-based techniques for pathogen detection: Retrospect,

recent advances, and future perspectives. Journal of Advanced Research, 50, 69–82.

https://doi.org/10.1016/j.jare.2022.10.011

Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F., & Collins, J. J. (2021). CRISPR-based

diagnostics. Nature Biomedical Engineering, 5(7), Article 7. https://doi.org/10.1038/s41551-021-

00760-7

Kanitchinda, S., Srisala, J., Suebsing, R., Prachumwat, A., & Chaijarasphong, T. (2020). CRISPR-Cas

fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and

specific detection of Enterocytozoon hepatopenaei. Biotechnology Reports, 27, e00485.

https://doi.org/10.1016/j.btre.2020.e00485

19

Electronic copy available at: https://ssrn.com/abstract=4815342

Khan, F. A., Pandupuspitasari, N. S., Chun-Jie, H., Ao, Z., Jamal, M., Zohaib, A., Khan, F. A., Hakim, M. R., &

ShuJun, Z. (2016). CRISPR/Cas9 therapeutics: A cure for cancer and other genetic diseases.

Oncotarget, 7(32), 52541–52552. https://doi.org/10.18632/oncotarget.9646

Kleppe, L., Fjelldal, P. G., Andersson, E., Hansen, T., Sanden, M., Bruvik, A., Skaftnesmo, K. O., Furmanek,

T., Kjærner-Semb, E., Crespo, D., Flavell, S., Pedersen, A. Ø., Vogelsang, P., Torsvik, A., Kvestad, K.

A., Olausson, S., Norberg, B., Schulz, R. W., Bogerd, J., … Wargelius, A. (2022). Full production

cycle performance of gene-edited, sterile Atlantic salmon—Growth, smoltification, welfare

indicators and fillet composition. Aquaculture, 560, 738456.

https://doi.org/10.1016/j.aquaculture.2022.738456

Kotob, M. H., Menanteau-Ledouble, S., Kumar, G., Abdelzaher, M., & El-Matbouli, M. (2016). The impact

of co-infections on fish: A review. Veterinary Research, 47(1), 98.

https://doi.org/10.1186/s13567-016-0383-4

Kumari, M., Lu, R.-M., Li, M.-C., Huang, J.-L., Hsu, F.-F., Ko, S.-H., Ke, F.-Y., Su, S.-C., Liang, K.-H., Yuan, J. P.-

Y., Chiang, H.-L., Sun, C.-P., Lee, I.-J., Li, W.-S., Hsieh, H.-P., Tao, M.-H., & Wu, H.-C. (2022). A critical

overview of current progress for COVID-19: Development of vaccines, antiviral drugs, and

therapeutic antibodies. Journal of Biomedical Science, 29(1), 68.

https://doi.org/10.1186/s12929-022-00852-9

Li, C., Lin, N., Feng, Z., Lin, M., Guan, B., Chen, K., Liang, W., Wang, Q., Li, M., You, Y., & Chen, Q. (2021).

CRISPR/Cas12a Based Rapid Molecular Detection of Acute Hepatopancreatic Necrosis Disease in

Shrimp. Frontiers in Veterinary Science, 8, 819681. https://doi.org/10.3389/fvets.2021.819681

Li, D., Li, X., Wang, Q., & Hao, Y. (2022). Advanced Techniques for the Intelligent Diagnosis of Fish

Diseases: A Review. Animals : An Open Access Journal from MDPI, 12(21), 2938.

https://doi.org/10.3390/ani12212938

20

Electronic copy available at: https://ssrn.com/abstract=4815342

Li, H., Cao, X., Chen, R., Guang, M., Xu, M., Wu, X., Yang, R., Lei, L., & Zhang, F. (2023). Rapid detection of

grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system.

Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1296038

Li, L., Li, S., Wu, N., Wu, J., Wang, G., Zhao, G., & Wang, J. (2019). HOLMESv2: A CRISPR-Cas12b-Assisted

Platform for Nucleic Acid Detection and DNA Methylation Quantitation. ACS Synthetic Biology,

8(10), 2228–2237. https://doi.org/10.1021/acssynbio.9b00209

Li, S.-Y., Cheng, Q.-X., Wang, J.-M., Li, X.-Y., Zhang, Z.-L., Gao, S., Cao, R.-B., Zhao, G.-P., & Wang, J. (2018).

CRISPR-Cas12a-assisted nucleic acid detection. Cell Discovery, 4(1), Article 1.

https://doi.org/10.1038/s41421-018-0028-z

Liang, J., Teng, P., Xiao, W., He, G., Song, Q., Zhang, Y., Peng, B., Li, G., Hu, L., Cao, D., & Tang, Y. (2021).

Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of

SARS-CoV-2 from clinical samples. Journal of Nanobiotechnology, 19, 273.

https://doi.org/10.1186/s12951-021-01021-0

Lin, Z., Lu, J., Wu, S., Lin, X., Zheng, L., Lou, Y., & Xiao, X. (2022). A novel detection method for the

pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex

RAA and CRISPR/Cas12a. Frontiers in Microbiology, 13.

https://www.frontiersin.org/articles/10.3389/fmicb.2022.973996

Liu, J., Li, Y., Cao, H., Yao, S., Hu, K., Zhao, Q., He, R., Zhu, N., Yu, X., Fang, S., & Huang, J. (2023).

Amplification-free CRISPR-Cas13a assay for detection of Taura syndrome virus. Aquaculture

Reports, 30, 101552. https://doi.org/10.1016/j.aqrep.2023.101552

Liu, S., Xie, T., Pei, X., Li, S., He, Y., Tong, Y., & Liu, G. (2023). CRISPR-Cas12a coupled with universal gold

nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection.

Sensors and Actuators. B, Chemical, 377, 133009. https://doi.org/10.1016/j.snb.2022.133009

21

Electronic copy available at: https://ssrn.com/abstract=4815342

Lou, J., Wang, B., Li, J., Ni, P., Jin, Y., Chen, S., Xi, Y., Zhang, R., & Duan, G. (2022). The CRISPR-Cas system

as a tool for diagnosing and treating infectious diseases. Molecular Biology Reports, 49(12),

11301–11311. https://doi.org/10.1007/s11033-022-07752-z

MacAulay, S., Ellison, A. R., Kille, P., & Cable, J. (2022). Moving towards improved surveillance and earlier

diagnosis of aquatic pathogens: From traditional methods to emerging technologies. Reviews in

Aquaculture, 14(4), 1813–1829. https://doi.org/10.1111/raq.12674

Mahas, A., Marsic, T., Lopez-Portillo Masson, M., Wang, Q., Aman, R., Zheng, C., Ali, Z., Alsanea, M., Al-

Qahtani, A., Ghanem, B., Alhamlan, F., & Mahfouz, M. (2022). Characterization of a thermostable

Cas13 enzyme for one-pot detection of SARS-CoV-2. Proceedings of the National Academy of

Sciences, 119(28), e2118260119. https://doi.org/10.1073/pnas.2118260119

Majid, S., Khan, M. S., Rashid, S., Niyaz, A., Farooq, R., Bhat, S. A., Wani, H. A., & Qureshi, W. (2021).

COVID-19: Diagnostics, Therapeutic Advances, and Vaccine Development. Current Clinical

Microbiology Reports, 8(3), 152–166. https://doi.org/10.1007/s40588-021-00157-9

Major, S. R., Harke, M. J., Cruz-Flores, R., Dhar, A. K., Bodnar, A. G., & Wanamaker, S. A. (2023). Rapid

Detection of DNA and RNA Shrimp Viruses Using CRISPR-Based Diagnostics. Applied and

Environmental Microbiology, 0(0), e02151-22. https://doi.org/10.1128/aem.02151-22

Makarova, K. S., & Koonin, E. V. (2015). Annotation and Classification of CRISPR-Cas Systems. Methods in

Molecular Biology (Clifton, N.J.), 1311, 47–75. https://doi.org/10.1007/978-1-4939-2687-9_4

Manessis, G., Gelasakis, A. I., & Bossis, I. (2022). Point-of-Care Diagnostics for Farm Animal Diseases:

From Biosensors to Integrated Lab-on-Chip Devices. Biosensors, 12(7), 455.

https://doi.org/10.3390/bios12070455

Maulu, S., Hasimuna, O. J., Haambiya, L. H., Monde, C., Musuka, C. G., Makorwa, T. H., Munganga, B. P.,

Phiri, K. J., & Nsekanabo, J. D. (2021). Climate Change Effects on Aquaculture Production:

22

Electronic copy available at: https://ssrn.com/abstract=4815342

 Sustainability Implications, Mitigation, and Adaptations. Frontiers in Sustainable Food Systems, 5.

https://www.frontiersin.org/articles/10.3389/fsufs.2021.609097

Morio, F., Lombardi, L., & Butler, G. (2020). The CRISPR toolbox in medical mycology: State of the art and

perspectives. PLoS Pathogens, 16(1), e1008201. https://doi.org/10.1371/journal.ppat.1008201

Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., Kellner, M. J.,

Tan, A. L., Paul, L. M., Parham, L. A., Garcia, K. F., Barnes, K. G., Chak, B., Mondini, A., Nogueira,

M. L., Isern, S., Michael, S. F., Lorenzana, I., Yozwiak, N. L., … Sabeti, P. C. (2018). Field-deployable

viral diagnostics using CRISPR-Cas13. Science (New York, N.Y.), 360(6387), 444–448.

https://doi.org/10.1126/science.aas8836

Naranitus, P., Aiamsa-At, P., Sukonta, T., Hannanta-Anan, P., & Chaijarasphong, T. (2022). Smartphone-

compatible, CRISPR-based platforms for sensitive detection of acute hepatopancreatic necrosis

disease in shrimp. Journal of Fish Diseases, 45(12), 1805–1816.

https://doi.org/10.1111/jfd.13702

Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia,

N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O’Connor, D. H., Gehrke, L., & Collins, J. J.

(2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059

Parsaeimehr, A., Ebirim, R. I., & Ozbay, G. (2022). CRISPR-Cas technology a new era in genomic

engineering. Biotechnology Reports, 34, e00731. https://doi.org/10.1016/j.btre.2022.e00731

Pulkkinen, K., Suomalainen, L.-R., Read, A. F., Ebert, D., Rintamäki, P., & Valtonen, E. T. (2010). Intensive

fish farming and the evolution of pathogen virulence: The case of columnaris disease in Finland.

Proceedings of the Royal Society B: Biological Sciences, 277(1681), 593–600.

https://doi.org/10.1098/rspb.2009.1659

23

Electronic copy available at: https://ssrn.com/abstract=4815342

Qi, Y., Li, K., Li, Y., Guo, D., Xu, J., Li, Y., & Gong, W. (2022). CRISPR-Based Diagnostics: A Potential Tool to

Address the Diagnostic Challenges of Tuberculosis. Pathogens, 11(10), 1211.

https://doi.org/10.3390/pathogens11101211

Quan, J., Langelier, C., Kuchta, A., Batson, J., Teyssier, N., Lyden, A., Caldera, S., McGeever, A., Dimitrov,

B., King, R., Wilheim, J., Murphy, M., Ares, L. P., Travisano, K. A., Sit, R., Amato, R.,

Mumbengegwi, D. R., Smith, J. L., Bennett, A., … Crawford, E. D. (2019). FLASH: A next-generation

CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. Nucleic Acids

Research, 47(14), e83. https://doi.org/10.1093/nar/gkz418

Quansah, E., Chen, Y., Yang, S., Wang, J., Sun, D., Zhao, Y., Chen, M., Yu, L., & Zhang, C. (2023). CRISPR-

Cas13 in malaria parasite: Diagnosis and prospective gene function identification. Frontiers in

Microbiology, 14, 1076947. https://doi.org/10.3389/fmicb.2023.1076947

Rong, F., Wang, H., Tang, X., Xing, J., Sheng, X., Chi, H., & Zhan, W. (2024). The development of RT-RPA

and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus

(IHNV). Journal of Virological Methods, 326, 114892.

https://doi.org/10.1016/j.jviromet.2024.114892

Shen, J., Zhou, X., Shan, Y., Yue, H., Huang, R., Hu, J., & Xing, D. (2020). Sensitive detection of a bacterial

pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.

Nature Communications, 11(1), Article 1. https://doi.org/10.1038/s41467-019-14135-9

Shinoda, H., Taguchi, Y., Nakagawa, R., Makino, A., Okazaki, S., Nakano, M., Muramoto, Y., Takahashi, C.,

Takahashi, I., Ando, J., Noda, T., Nureki, O., Nishimasu, H., & Watanabe, R. (2021). Amplification-

free RNA detection with CRISPR–Cas13. Communications Biology, 4(1), Article 1.

https://doi.org/10.1038/s42003-021-02001-8

24

Electronic copy available at: https://ssrn.com/abstract=4815342

Shyam, K. U., Kim, H.-J., Kole, S., Oh, M.-J., Kim, C.-S., Kim, D.-H., & Kim, W.-S. (2022). Antibody-based

lateral flow chromatographic assays for detecting fish and shrimp pathogens: A technical review.

Aquaculture, 558, 738345. https://doi.org/10.1016/j.aquaculture.2022.738345

Singh, M., Bindal, G., Misra, C. S., & Rath, D. (2022). The era of Cas12 and Cas13 CRISPR-based disease

diagnosis. Critical Reviews in Microbiology, 48(6), 714–729.

https://doi.org/10.1080/1040841X.2021.2025041

Sukonta, T., Senapin, S., Meemetta, W., & Chaijarasphong, T. (2022). CRISPR-based platform for rapid,

sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates

calcarifer). Journal of Fish Diseases, 45(1), 107–120. https://doi.org/10.1111/jfd.13541

Sukonta, T., Senapin, S., Taengphu, S., Hannanta-anan, P., Kitthamarat, M., Aiamsa-at, P., &

Chaijarasphong, T. (2022). An RT-RPA-Cas12a platform for rapid and sensitive detection of tilapia

lake virus. Aquaculture, 560, 738538. https://doi.org/10.1016/j.aquaculture.2022.738538

Sullivan, T. J., Dhar, A. K., Cruz-Flores, R., & Bodnar, A. G. (2019). Rapid, CRISPR-Based, Field-Deployable

Detection Of White Spot Syndrome Virus In Shrimp. Scientific Reports, 9, 19702.

https://doi.org/10.1038/s41598-019-56170-y

Suminda, G. G. D., Bhandari, S., Won, Y., Goutam, U., Kanth Pulicherla, K., Son, Y.-O., & Ghosh, M. (2022).

High-throughput sequencing technologies in the detection of livestock pathogens, diagnosis, and

zoonotic surveillance. Computational and Structural Biotechnology Journal, 20, 5378–5392.

https://doi.org/10.1016/j.csbj.2022.09.028

Sun, Y., Yang, C., Jiang, X., Zhang, P., Chen, S., Su, F., Wang, H., Liu, W., He, X., Chen, L., Man, B., & Li, Z.

(2023). High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple

with stabilized graphene field-effect transistor. Biosensors and Bioelectronics, 222, 114979.

https://doi.org/10.1016/j.bios.2022.114979

25

Electronic copy available at: https://ssrn.com/abstract=4815342

Tao, Z., Wang, B., Cui, Q., Wang, P., Dzantiev, B. B., Wan, Y., Wu, J., & Yang, Z. (2023). A signal-off

Cas14a1-based platform for highly specific detection of methicillin-resistant Staphylococcus

aureus. Analytica Chimica Acta, 1256, 341154. https://doi.org/10.1016/j.aca.2023.341154

Timilsina, A., Adhikari, K., Yadav, A. K., Joshi, P., Ramena, G., & Bohara, K. (2023). Effects of microplastics

and nanoplastics in shrimp: Mechanisms of plastic particle and contaminant distribution and

subsequent effects after uptake. Science of The Total Environment, 894, 164999.

https://doi.org/10.1016/j.scitotenv.2023.164999

Wang, L., He, F., Chen, X., He, K., Bai, L., Wang, Q., Zhang, F., & Xu, X. (2022). A CRISPR/Cas12a-based

label-free fluorescent method for visual signal output. Sensors and Actuators B: Chemical, 370,

132368. https://doi.org/10.1016/j.snb.2022.132368

Wang, P., Guo, B., Zhang, X., Wang, Y., Yang, G., Shen, H., Gao, S., & Zhang, L. (2023). One-Pot Molecular

Diagnosis of Acute Hepatopancreatic Necrosis Disease by Recombinase Polymerase

Amplification and CRISPR/Cas12a with Specially Designed crRNA. Journal of Agricultural and

Food Chemistry, 71(16), 6490–6498. https://doi.org/10.1021/acs.jafc.2c08689

Wang, P., Yang, L., Guo, B., Shang, Z., Gao, S., & Zhang, X. (2023). Highly sensitive detection of white spot

syndrome virus with an RPA-CRISPR combined one-pot method. Aquaculture, 567, 739296.

https://doi.org/10.1016/j.aquaculture.2023.739296

Wang, P., Zhang, X., Shen, H., Lu, Q., Li, B., Liu, X., & Gao, S. (2023). A one-pot RPA-CRISPR detection

method for point-of-care testing of Enterocytozoon hepatopenaei infection in shrimp. Sensors

and Actuators B: Chemical, 374, 132853. https://doi.org/10.1016/j.snb.2022.132853

Wang, Q., Mao, C., Lei, L., Yan, B., Yuan, J., Guo, Y., Li, T., Xiong, X., Cao, X., Huang, J., Han, J., Yu, K., &

Zhou, B. (2022). Antibiotic resistance genes and their links with bacteria and environmental

factors in three predominant freshwater aquaculture modes. Ecotoxicology and Environmental

Safety, 241, 113832. https://doi.org/10.1016/j.ecoenv.2022.113832

26

Electronic copy available at: https://ssrn.com/abstract=4815342

Wang, Y., Hou, Y., Liu, X., Lin, N., Dong, Y., Liu, F., Xia, W., Zhao, Y., Xing, W., Chen, J., & Chen, C. (2023).

Rapid visual nucleic acid detection of Vibrio alginolyticus by recombinase polymerase

amplification combined with CRISPR/Cas13a. World Journal of Microbiology and Biotechnology,

40(2), 51. https://doi.org/10.1007/s11274-023-03847-2

WOAH. (n.d.). PRINCIPLES AND METHODS OF VALIDATION OF DIAGNOSTIC ASSAYS FOR INFECTIOUS

DISEASES.

https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/1.01.06_VALIDATION.pdf

WOAH. (2023, January 2). Aquatic Manual Online Access. WOAH - World Organisation for Animal Health.

https://www.woah.org/en/what-we-do/standards/codes-and-manuals/aquatic-manual-online-

access/

Xiao, X., Lin, Z., Huang, X., Lu, J., Zhou, Y., Zheng, L., & Lou, Y. (2021). Rapid and Sensitive Detection of

Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay.

Frontiers in Microbiology, 12. https://www.frontiersin.org/articles/10.3389/fmicb.2021.767315

Yang, S., & Rothman, R. E. (2004). PCR-based diagnostics for infectious diseases: Uses, limitations, and

future applications in acute-care settings. The Lancet. Infectious Diseases, 4(6), 337–348.

https://doi.org/10.1016/S1473-3099(04)01044-8

Yue, K., & Shen, Y. (2022). An overview of disruptive technologies for aquaculture. Aquaculture and

Fisheries, 7(2), 111–120. https://doi.org/10.1016/j.aaf.2021.04.009

Zhang, C., Tao, Z., Ye, H., Wang, P., Jiang, M., Benard, K., Li, W., & Yan, X. (2024). Development and

validation of a CRISPR/Cas12a-based platform for rapid and sensitive detection of the large

yellow croaker iridovirus. Aquaculture, 584, 740658.

https://doi.org/10.1016/j.aquaculture.2024.740658

Zhang, W. S., Pan, J., Li, F., Zhu, M., Xu, M., Zhu, H., Yu, Y., & Su, G. (2021). Reverse Transcription

Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly

27

Electronic copy available at: https://ssrn.com/abstract=4815342

 Sensitive Colorimetric SARS-CoV-2 Detection. Analytical Chemistry, 93(8), 4126–4133.

https://doi.org/10.1021/acs.analchem.1c00013

Zhong, Z., Niu, P., Wang, M., Huang, G., Xu, S., Sun, Y., Xu, X., Hou, Y., Sun, X., Yan, Y., & Wang, H. (2016).

Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and

more muscular cells in common carp. Scientific Reports, 6(1), Article 1.

https://doi.org/10.1038/srep22953

28

Electronic copy available at: https://ssrn.com/abstract=4815342