The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.

/pdf/crispr-based-diagnostics-4r70xy687r.pdf

CRISPR-based diagnostics.

For infectious diseases, rapid and accurate identification of the pathogen is critical for effective management and treatment, but diagnosis remains challenging, particularly in resource-limited areas. Methods that accurately detect pathogen nucleic acids can provide robust, accurate, rapid, and ultrasensitive technologies for point-of-care diagnosis of pathogens, and thus yield information that is invaluable for disease management and treatment. Several technologies, mostly PCR-based, have been employed for pathogen detection; however, these require expensive reagents and equipment, and skilled personnel. CRISPR/Cas systems have been used for genome editing, based on their ability to accurately recognize and cleave specific DNA and RNA sequences. Moreover, following recognition of the target sequence, certain CRISPR/Cas systems including orthologues of Cas13, Cas12a, and Cas14 exhibit collateral nonspecific catalytic activities that can be employed for nucleic acid detection, for example by degradation of a labeled nucleic acid to produce a fluorescent signal. CRISPR/Cas systems are amenable to multiplexing, thereby enabling a single diagnostic test to identify multiple targets down to attomolar (10-18 mol/L) concentrations of target molecules. Developing devices that couple CRISPR/Cas with lateral flow systems may allow inexpensive, accurate, highly sensitive, in-field deployable diagnostics. These sensors have myriad applications, from human health to agriculture. In this review, we discuss the recent advances in the field of CRISPR-based biosensing technologies and highlight insights of their potential use in a myriad of applications.

Nucleic Acid Detection Using CRISPR/Cas Biosensing Technologies.

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

/pdf/crispr-cas-a-nobel-prize-award-winning-precise-genome-3hxgsbr1b6.pdf

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement.

Ideal methods for detecting pathogens should be sensitive, specific, rapid, cost-effective and instrument-free. Conventional nucleic acid pathogen detection strategies, mostly PCR-based techniques, have various limitations, such as expensive equipment, reagents and skilled performance. Recently, CRISPR/Cas-based methods have burst onto the scene, with the potential to power the pathogen detection field. Here we introduce these unique methods and discuss its hurdles and promises.

https://www.tandfonline.com/doi/pdf/10.1080/22221751.2020.1793689

Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods.

https://doi.org/10.1016/j.bios.2022.113994

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Rapid and simple-to-use diagnostic methods for tuberculosis are urgently needed. Recent development has unveiled the diagnostic power of the CRISPR system in the detection of viral infections. Howe...

https://www.tandfonline.com/doi/pdf/10.1080/22221751.2019.1664939

CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis

Objective: To evaluate the effectiveness of Xpert MTB/RIF in patients with multidrug-resistant tuberculosis (MDR-TB). Methods: Seventy-five patients with MDR-TB were enrolled in this prospective cohort study and were divided into two groups. The observation group were given standardized anti-MDR-TB treatment regimen (6ZAmLfxPtoCs/18ZLfxPtoCs) immediately when they had two positive sputum Xpert MTB/RIF results of RIF resistance. The control group were not given standardized anti-MDR-TB regimen until culture-based drug-susceptibility testing suggested MDR-TB. Treatment effect index, foci absorption, conversion of sputum, treatment outcomes, and adverse reactions were observed. Fisher's exact test and chi-square test were used to compare the differences between groups. Results: Treatment effect index of the observation group significantly out-performed the control group (24/34, 70.6% vs. 17/38, 44.7%, p = 0.027). At the 6th month of treatment course, observation group achieved significantly higher conversion (28/34, 82.3% vs. 23/38, 60.5%, p = 0.042). The foci absorption, cavity change, conversion at the 24th month of course, or treatment outcome between two groups were not statistically different. Conclusion: Xpert MTB/RIF helps MDR-TB patients to start rational treatment regimen earlier and reach earlier sputum conversion.

Xpert MTB/RIF Improves the Prognosis of Multidrug-Resistant Tuberculosis Patients Through Faster Diagnosis and Earlier Targeted Treatment: A Prospective Cohort Study.

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CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis

Xpert MTB/RIF Improves the Prognosis of Multidrug-Resistant Tuberculosis Patients Through Faster Diagnosis and Earlier Targeted Treatment: A Prospective Cohort Study.