CRISPR/Cas Biosensors Address Sensitivity and Specificity Challenges in Detection

Detecting and monitoring nucleic acid contaminants and chemical contaminants including toxic small molecules is essential for safeguarding vital aspects of human life such as food production and our environment.

In a recent review published in Environmental Technology & Innovation, researchers discussed the mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)-based biosensors, and the versatility and advantages of using CRISPR/Cas biosensors to detect contaminants.

​​​​​​​Study: CRISPR/Cas12a-based biosensors for environmental monitoring and diagnostics. Image Credit: Marcin Janiec/Shutterstock.com​​​​​​​Study: CRISPR/Cas12a-based biosensors for environmental monitoring and diagnostics. Image Credit: Marcin Janiec/Shutterstock.com

Background

Pollutants entering the environment from various spheres of human life, such as industries, agriculture, vehicles, and urban life increase the risk of clean water contamination.

Water ecology is intrinsically linked to numerous other ecological factors, such as flood regulation, food production, soil formation, nutrient cycling, and habitat availability.

Nucleic acid contaminants in water have been associated with factors such as ecological imbalance and the transmission of diseases. In contrast, non-nucleic acid contaminants such as heavy metals, agrochemicals, antibiotics, and plastics have been prevalent in water ecosystems, especially near urban areas.

While traditional diagnostic and environmental monitoring systems such as chromatography and spectroscopy and even more modern methods such as metagenomics have played important roles in the detection of contaminants, portability, sensitivity, and specificity continue to present challenges.

CRISPR/Cas technology

CRISPR/Cas technology applications have surpassed genome editing, and biosensors using the CRISPR/Cas technology are now being used for environmental monitoring. The CRISPR/Cas12a system has been widely used to detect nucleic acids such as genes, genetic markers, and pathogens.

The ability of Cas12a to target specific deoxyribonucleic acid (DNA) sequences can be utilized and modified to elicit a molecular response upon target sequence binding, which can then be used to detect nucleic acids with precision and sensitivity.

Furthermore, the ability of the Cas12a enzyme to cleave molecules has also been utilized to detect non-nucleic acid contaminants such as small molecules, proteins, and various environmental indicators.

The present review discussed the innovative methods in which the CRISPR/Cas12a system has been applied to build biosensors to detect nucleic acids and non-nucleic acid contaminants in water.

Conventional methods

The researchers discussed some of the conventional methods used to detect non-nucleic acid contaminants, such as molecules of agrochemical and industrial origin. These methods included spectroscopic methods, sensor deployment strategies, microfluidic devices, biosensors, and traditional instrument-based methods.

Fourier-transform infrared (FTIR) spectroscopy-based detection methods have been used to identify pesticide contaminants. In contrast, another method has used carbon nanotubes coupled with high-performance liquid chromatography and diode array detection for the same.

Detection of industrial contaminants has conventionally been conducted through atomic absorption spectroscopy and mass spectrometry. Heavy metal detection has also been conducted through colorimetric and attenuated total reflectance-FTIR methods.

Detecting nucleic acid contaminants has depended largely on polymerase chain reaction (PCR)-based methods, culturing and biochemical analyses, and immunoassays.

Non-culturable organisms and nucleic acid contaminants have also been detected through whole-genome or 16S ribonucleic acid (RNA) sequencing or metagenomic methods.

Electrochemical biosensors, using gold nanoparticles, iron oxide, and graphene oxide, have also been used to detect phenolic substances such as hydroquinone and catechol.

Boron-doped diamond electrodes and a magneto-enzyme immunoassay have also been used to detect herbicides such as atrazine in the environment.

CRISPR/Cas12a biosensors

The endonucleolytic activity of the Cas12a enzyme has made it valuable for genetic engineering and the detection of nucleic acids. The researchers briefly described the mechanism through which Cas12a cleaves DNA or RNA targets and the steps involved in developing CRISPR/Cas12a biosensors.

A critical part of CRISPR/Cas12a biosensors is the guide RNA, which is complementary to the RNA or DNA target region and the choice of which impacts the efficiency and specificity of the CRISPR/Cas12a biosensor.

Therefore, considerable effort is invested in optimizing the design of the guide RNA. Once the CRISPR/Cas12a system binds to the target region with the help of the guide RNA, the conformational changes that ensue activate the endonucleolytic activity.

The review discussed various systems in which detection methods such as fluorescence, colorimetry, and electrochemical signals are coupled with the CRISPR/Cas12a system to detect the reporter molecules released when the Cas12a cleaves the target nucleic acids.

The detection of small molecules using the CRISPR/Cas12a system involves the use of allosteric transcription factors (ATF). ATF binds to the target DNA without small molecule contaminants and prevents CRISPR/Cas12a from binding to the DNA.

The presence of small molecules is detected when they bind to the allosteric transcription factors, releasing them from the target DNA, and allowing CRISPR/Cas12a to bind to the DNA and cleave it, releasing reporter molecules.

The researchers also provided a detailed summary of various studies that have utilized the CRISPR/Cas12a system to detect non-nucleic acid contaminants, including methods where the CRISPR/Cas12a system is coupled with colorimetric or electrochemical detection methods.

Conclusions

Overall, the review provided a comprehensive view of the advances in biosensor technologies to detect nucleic acids and non-nucleic acid contaminants in water, ranging from early and conventional detection methods to the recent technological achievement of CRISPR/Cas12a-based biosensors.

Combining the CRISPR/Cas12a technology with molecular methods, nanotechnology, and electrochemical detection techniques provides a more accurate and efficient method of detecting contaminants in water, ensuring a safer environment.

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