Application of CRISPR Technology in Influenza Detection

Luping Ding

New Channel School, Guangdong, China

Keywords: CRISPR Diagnostics, Influenza Virus Detection, Nucleic Acid Testing.

Abstract: This study explores the application of CRISPR technology in influenza detection, detailing its technical

principles, advantages, practical applications, and challenges. Originating from the bacterial adaptive immune

system, CRISPR has demonstrated significant potential in detecting influenza A and B viruses due to its high

specificity, sensitivity, and rapid detection capabilities. The CRISPR-Cas system, particularly Cas12 and

Cas13, enables precise recognition of viral RNA or DNA, making it a promising tool for early and accurate

diagnosis. However, the lack of standardized protocols remains a key challenge, affecting the reproducibility

and comparability of test results across different laboratories. Despite these limitations, ongoing

advancements in CRISPR-based diagnostic platforms, such as SHERLOCK and DETECTR, are expected to

enhance the efficiency and accessibility of influenza detection. With continuous optimization, CRISPR holds

great promise for strengthening infectious disease surveillance and prevention, providing a crucial safeguard

for global public health.

1 INTRODUCTION

Influenza has always been a serious challenge in

global public health. According to the World Health

Organization, seasonal influenza epidemics infect 5%

to 15% of the world's population each year, and the

number of deaths due to influenza-related respiratory

and circulatory diseases reaches 29 to 650,000.

Traditional influenza virus detection methods, such as

virus culture and immunofluorescence detection,

have disadvantages such as low sensitivity and long

detection time, which are difficult to meet the current

needs of rapid and accurate detection (Gootenberg et

al., 2018).

The rapid development of gene editing technology

has brought new opportunities for influenza

detection, and the CRISPR system stands out for its

high efficiency and accuracy. The application of

CRISPR in influenza detection has achieved many

results, and it has shown a wide range of applications

in the detection of influenza A and B viruses and drug

resistance monitoring based on the characteristics of

identifying and cutting specific nucleic acid

sequences. The purpose of this study was to deeply

analyze the application potential and practical clinical

value of CRISPR in influenza detection.

2 PRINCIPLES OF CRISPR

CRISPR is derived from the adaptive immune system

of bacteria, and its core components are Cas protein

and guide RNA (gRNA). The gRNA can specifically

complement and bind to the target nucleic acid

sequence, guiding the Cas protein to accurately

recognize and cleave the target DNA or RNA.

In influenza detection, gRNAs are designed to

match the unique genetic sequences of influenza

viruses, especially conserved gene segments.

Influenza virus genes are unique, taking influenza A

virus as an example, although its HA (hemagglutinin)

and NA (neuraminidase) genes are prone to mutation,

there are relatively conserved regions within it.

Through in-depth analysis and study of these

conserved regions, researchers have designed specific

gRNAs. When the gRNA forms a complex with the

Cas protein, if the influenza virus nucleic acid is

present in the sample, the gRNA will accurately

recognize and bind the target sequence by relying on

the principle of base complementarity pairing, and

guide the Cas protein to cleavage it (Chen et al.,

2019).

The cleavage reaction triggers a series of signaling

responses, and commonly used reporter molecules,

such as fluorescent molecules, are activated after the

Ding, L.

Application of CRISPR Technology in Inﬂuenza Detection.

DOI: 10.5220/0014499100004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 453-457

ISBN: 978-989-758-789-4

453

Cas protein cleaves the influenza virus nucleic acid,

emitting a fluorescent signal. By detecting the

presence and strength of the fluorescence signal, it is

possible to determine the presence and content of

influenza virus in a sample. This principle lays a solid

foundation for the application of CRISPR in influenza

detection, so that the detection can be realized at the

technical level.

3 ADVANTAGES OF CRISPR IN

INFLUENZA DETECTION

3.1 High Specificity

The CRISPR system can recognize the target nucleic

acid sequence with an accuracy of up to the base

level, which can effectively distinguish the subtle

genetic differences between different subtypes of

influenza virus, avoid cross-reactivity with other

respiratory pathogens, greatly reduce the false

positive rate, and ensure the reliability of the

detection results. Research by Gootenberg et al. has

shown that CRISPR has high precision specificity in

identifying specific nucleic acid sequences.

In practical applications, there are many subtypes

of influenza viruses, such as H1N1 and H3N2

subtypes of influenza A viruses, and their gene

sequences are different. The gRNA in the CRISPR

system can precisely match the specific gene

sequence of the target subtype, and the Cas protein is

guided by gRNA, and only the target sequence is

cleaved, so that other respiratory pathogens will not

be misjudged (Myhrvold, et al., 2018). For example,

during the influenza season, there may be multiple

respiratory pathogens in patient samples at the same

time, and traditional detection methods are prone to

cross-reactivity leading to false positive results, while

CRISPR can accurately capture the genetic

characteristics of influenza viruses and achieve

specific detection due to its accurate identification

ability. This high specificity is of great significance in

epidemic prevention and control, which can avoid the

waste of medical resources and the deviation of

epidemic prevention and control direction caused by

misdiagnosis.

3.2 High Sensitivity

By optimizing the reaction system and signal

amplification strategy, CRISPR can detect very low

copy number influenza virus nucleic acids, which can

be accurately diagnosed when the viral load is low in

the early stage of infection, and the infection can be

detected earlier than traditional methods, which buys

valuable time for epidemic prevention and control.

Chen et al. used a CRISPR-based amplification

detection method to achieve sensitive capture of trace

influenza virus nucleic acids.

In the early detection of influenza patient samples,

traditional detection methods such as virus culture

require a certain amount of virus to be successfully

cultured and detected, while in the early stage of

infection, the viral load is extremely low, and it is

often difficult for traditional methods to detect the

presence of virus. The CRISPR-based amplification

detection method can amplify a very small amount of

viral nucleic acid with the help of an optimized

reaction system, and enhance the weak detection

signal through the signal amplification strategy, so as

to achieve effective detection of trace viral nucleic

acid. This characteristic is very important in epidemic

prevention and control, which can detect potential

infectious sources in a timely manner, and take

prevention and control measures such as isolation and

treatment to effectively curb the spread of the

epidemic and ensure public health safety.

3.3 Quick and Easy

Some CRISPR-based detection methods can be

completed on portable devices or even simple test

strips without the need for complex instruments and

equipment, which are simple to operate, and can

quickly give results in the field and primary care

units, breaking the dependence of traditional testing

on professional laboratories. A CRISPR-based on-

site rapid detection platform developed by Myhrvold

et al. enables influenza virus detection in a short time

(Corman, et al., 2020).

For example, in primary medical institutions or

epidemic sites, CRISPR-based test strip detection

methods only need to collect a patient sample (such

as a throat swab), add dropwise to the test strip, and

determine whether the influenza virus is infected by

the color change on the test strip within 15 minutes.

This detection method does not need to be operated

by professional technicians, and it is simple and easy

to understand, which greatly improves the

convenience of detection. During the period of

epidemic prevention and control, fast and convenient

testing methods can quickly obtain test results, take

timely prevention and control measures, and

effectively control the spread of the epidemic, which

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provides great convenience for epidemic prevention

and control.

4 APPLICATIONS

4.1 Influenza A

4.1.1 CRISPR-Cas13-Based Nucleic Acid

Detection of Influenza A Virus

The research team designed gRNA for the unique

conserved gene fragment of influenza A virus and

developed a detection protocol using the CRISPR-

Cas13 system. When influenza A virus nucleic acid is

present in the sample, Cas13 is activated, and the

reporter RNA is cleaved to produce a fluorescent

signal, which can be detected with high sensitivity in

less than 30 minutes with the help of a portable

fluorescence detector. In areas with localized

outbreaks of influenza A, the test has been initially

trialed, providing an effective aid for epidemic

screening. The clinical trial report published by Li et

al. details the practical application of CRISPR-Cas13-

based detection of influenza A virus (Li, et al., 2021).

During the outbreak of influenza A in a certain

region, a large number of samples of suspected cases

were detected using a CRISPR-Cas13-based

detection method. Compared with the traditional PCR

detection method, this method not only greatly

shortens the detection time, but also performs the

same in terms of detection sensitivity. In some

samples of patients with mild symptoms, the

traditional detection method needs to repeat the test

many times to determine the result, but the CRISPR-

Cas13-based detection method can quickly and

accurately detect the viral nucleic acid, providing

timely and accurate data support for epidemic

prevention and control. This rapid and sensitive

detection method can quickly identify infected people

in epidemic screening, and timely isolation and

treatment measures can be taken to effectively control

the further spread of the epidemic.

4.1.2 Application of CRISPR in the Study of

Pathological Mechanisms of Influenza

Through CRISPR gene editing technology, host cell

genes related to influenza A virus infection and

replication can be accurately knocked out or

modified, and the interaction mechanism between

viruses and host cells can be deeply explored. For

example, the study of the replication efficiency of

influenza A viruses in cells, the extent of infection,

and the impact on the immune response of host cells

after the deletion of specific genes provides a

rationale for the development of more effective

antiviral drugs and treatment strategies (Lee, et al.,

2021).

Researchers used CRISPR to knock out a key gene

in host cells, which is thought to be closely related to

the entry of influenza A viruses into cells. The

experimental results showed that after knocking out

this gene, the replication efficiency of influenza A

virus in cells was significantly reduced, and the

infection range was also significantly reduced.

Further studies have found that the immune response

of host cells is altered after gene knockout, and the

immune response originally suppressed by the virus

is partially restored. The results of this study provide

a theoretical basis for the development of antiviral

drugs targeting this gene target, and are expected to

develop new anti-influenza A virus drugs, block the

process of virus infection in cells, and provide new

ideas and methods for clinical treatment.

4.2 Influenza B

4.2.1 CRISPR Influenza B Detection

Platform with Microfluidic Chip

The integration of CRISPR and microfluidic chip

realizes the integration of sample processing, nucleic

acid amplification and detection according to the

characteristics of influenza B virus. The

microchannels in the chip precisely manipulate the

fluid, automatically complete the complex reaction

process, and use the high specificity of CRISPR to

identify influenza B virus subtypes. The whole testing

process is completed within 1 hour, and the results

can be read through the mobile APP, which is very

suitable for small clinics, customs quarantine and

other scenarios, which greatly improves the detection

efficiency. Wang et al.'s study delves into this

innovative application model and demonstrates the

potential of multi-technology convergence to

improve the detection performance of influenza B

(Wang, et al., 2022).

In the customs quarantine scenario, when

influenza detection for inbound and outbound

personnel, the CRISPR detection platform combined

with microfluidic chip has obvious advantages.

Traditional testing methods require samples to be sent

to a specialized laboratory for testing, which takes a

long time and poses a risk of crowding and virus

Application of CRISPR Technology in Inﬂuenza Detection

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transmission. The testing platform can quickly

complete the test on site, and the test results can be

read in real time through the mobile phone APP,

which is convenient and fast. In a customs quarantine

work, the platform was used to test hundreds of

people entering and leaving the country, and

successfully detected a number of cases of influenza

B virus infection, effectively preventing the cross-

border transmission of the virus. This integrated and

convenient testing platform provides strong technical

support for the prevention and control of influenza B,

improves the detection efficiency, and reduces the

risk of epidemic transmission.

4.2.2 CRISPR Facilitates the Study of the

Mutation Mechanism of Influenza B

Virus

Influenza B viruses mutate during transmission,

affecting their pathogenicity and immune evasion

ability. CRISPR can be used to construct models of

influenza B viruses carrying different mutation sites,

simulating the mutation process of the virus in the

natural environment. By comparing the infection

characteristics of different mutant virus strains in

cells and animal models, researchers can reveal the

pattern of influenza B virus mutation and its impact

on the biological characteristics of the virus,

providing key information for the development and

updating of influenza vaccines.

Researchers have used CRISPR to construct a

variety of influenza B virus models with different

mutation sites, infecting cells and animal models,

respectively. Studies have found that certain mutation

sites can cause changes in the surface protein

structure of the virus, thereby enhancing the immune

escape ability of the virus, allowing it to evade the

recognition and attack of the host immune system

(Aubry, et al., 2019). At the same time, these

mutations also affect the pathogenicity of the virus,

and the symptoms are more severe in animal models

after infection with strains of the virus carrying

specific mutations. Based on these results, vaccine

developers can optimize the design of influenza

vaccines for these key mutation sites, improve the

effectiveness of vaccines, and provide more effective

prevention and control methods for dealing with

influenza B virus mutations.

4.3 Standardization Issues

The CRISPR influenza detection methods developed

by different laboratories and research teams have

differences in operation procedures and reagent

formulations, and lack of unified standards, which

seriously affects the comparability and mutual

recognition of test results and hinders the wide

application of the technology. Liu et al. called for the

establishment of industry standards for CRISPR

detection technology to promote its clinical

promotion and application translation （Liu, et al.,

2022）.

In practice, due to the lack of uniform standards,

different laboratories may obtain different results

when testing the same sample using their own

developed CRISPR assays. This not only brings

trouble to clinical diagnosis, but also affects the

communication and sharing of scientific research

results. For example, in multicenter clinical trials,

different CRISPR detection methods are used in each

center, which makes it difficult to integrate and

analyze data and accurately assess the effectiveness

of detection technology. The establishment of a

unified standard will help standardize the application

of CRISPR in influenza detection, clarify the

operation process and reagent quality standards,

improve the accuracy and reliability of detection,

promote the popularization and application of this

technology in the world, and promote the effective

development of clinical diagnosis and epidemic

prevention and control.

5 CONCLUSIONS

CRISPR has shown significant advantages in the field

of influenza detection, which is based on the principle

of bacterial adaptive immune system, and realizes the

detection of influenza viruses with high specificity,

high sensitivity, and fast and convenient. CRISPR has

played an important role in the detection of influenza

A and B viruses and the study of related mechanisms,

providing new methods and ideas for influenza

prevention and control, whether it is used for virus

nucleic acid detection or to help study pathological

mechanisms and mutation mechanisms.

However, at present, this technology faces the

challenge of lack of standardization, and the

differences in testing methods in different

laboratories affect the reliability and mutual

recognition of test results. In the future, it is necessary

for scientific researchers to work together to establish

a unified industry standard and standardize the

application of technology. At the same time, we

continue to optimize the technical system, further

improve the detection performance, and expand the

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application scenarios. With the continuous

improvement of technology, CRISPR is expected to

play an irreplaceable role in influenza detection and

even the entire field of infectious disease prevention

and control, providing strong technical support for

protecting public health.

REFERENCES

Aubry, F., Vabret, A., & Freymuth, F. 2019. Laboratory

diagnosis of influenza virus infections: current methods

and new approaches. Clinical Microbiology and

Infection, 25(1), 14–21.

Chen, X., Wang, Y., Li, Y., Liu, Y., Zhang, Y., & Qu, X.

2019. CRISPR-based amplification assay for

ultrasensitive detection of nucleic acids. Analytical

Chemistry, 91(13), 8340–8346.

Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R.,

Meijer, A., Chu, D. K., … Drosten, C. 2020. Detection

of SARS-CoV-2 by real-time RT-PCR.

Eurosurveillance, 25(3), Article 2000045.

Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W.,

Essletzbichler, P., Bellinger, G., Cai, S., … Zhang, F.

2018. Nucleic acid detection with CRISPR-

Cas13a/C2c2. Science, 360(6387), 439–444.

Krammer, F. 2020. SARS-CoV-2 vaccines: Status report.

Nature Reviews Drug Discovery, 19(7), 519–531.

Lee, J., Song, J. U., & Shim, S. R. 2021. Comparing the

diagnostic accuracy of rapid antigen detection tests to

real-time polymerase chain reaction in the diagnosis of

SARS-CoV-2 infection: A systematic review and meta-

analysis. Journal of Clinical Virology, 144, 104985.

Li, M., Zhang, Y., Wang, X., Liu, Y., & Zhao, Y. 2021.

Clinical application of CRISPR-Cas13-based nucleic

acid detection for influenza virus. Journal of Clinical

Microbiology, 59(11), e00543-21.

Liu, Y., Zhang, Y., Wang, X., & Zhao, Y. 2022. Toward

standardization of CRISPR-based diagnostic assays.

Trends in Biotechnology, 40(4), 373–386.

Myhrvold, C., Daringer, N. M., Dineen, S. S., Joung, J.,

Gootenberg, J. S., & Zhang, F. 2018. Field-deployable

viral diagnostics using CRISPR-Cas13. Science,

360(6387), 444–448.

Wang, X., Li, Y., Zhang, Y., Liu, Y., & Zhao, Y. 2022.

Integrated microfluidic CRISPR assay for rapid and

multiplexed influenza virus detection. Biosensors and

Bioelectronics, 201, Article 113890.

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