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.

The global burden of invasive fungal infections (IFIs) has shown an upsurge in recent years due to the higher load of immunocompromised patients suffering from various diseases. The role of early and accurate diagnosis in the aggressive containment of the fungal infection at the initial stages becomes crucial thus, preventing the development of a life-threatening situation. With the changing demands of clinical mycology, the field of fungal diagnostics has evolved and come a long way from traditional methods of microscopy and culturing to more advanced non-culture-based tools. With the advent of more powerful approaches such as novel PCR assays, T2 Candida, microfluidic chip technology, next generation sequencing, new generation biosensors, nanotechnology-based tools, artificial intelligence-based models, the face of fungal diagnostics is constantly changing for the better. All these advances have been reviewed here giving the latest update to our readers in the most orderly flow.A detailed literature survey was conducted by the team followed by data collection, pertinent data extraction, in-depth analysis, and composing the various sub-sections and the final review. The review is unique in its kind as it discusses the advances in molecular methods; advances in serology-based methods; advances in biosensor technology; and advances in machine learning-based models, all under one roof. To the best of our knowledge, there has been no review covering all of these fields (especially biosensor technology and machine learning using artificial intelligence) with relevance to invasive fungal infections.The review will undoubtedly assist in updating the scientific community's understanding of the most recent advancements that are on the horizon and that may be implemented as adjuncts to the traditional diagnostic algorithms. 

/pdf/diagnosis-of-invasive-fungal-infections-challenges-and-2sfm74r4.pdf

Diagnosis of invasive fungal infections: challenges and recent developments

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

Dear Editors, Visceral leishmaniasis, histoplasmosis, and talaromycosis are infec‐ tions caused by Leishmania, Histoplasma, and Talaromyces marneffei, respectively. These infections have similar clinical manifestations and share similarities in bone marrow microscopic examinations, as they are located in mononuclear macrophages and have similar shapes and sizes. The gold standards for the diagnosis of these diseases are still direct microscopic examination and culture. However, culture meth‐ ods have low sensitivity1 and can be time-consuming. Microscopic examination relies on physician experience and subjective judgment.2 Other diagnostic approaches, such as qPCR and serologic antigen or antibody detection, require assumptions about the suspected patho‐ gen. As an alternative, rapid metagenomic next-generation sequenc‐ ing (mNGS) offers an objective tool for diagnosis. We report 5 cases with similar clinical manifestations and bone marrow histopathologic findings, presenting challenges to physicians. The causative agents of these cases of visceral leishmaniasis, histoplasmosis, and talaromy‐ cosis were identified by mNGS using both blood and bone marrow samples. Four patients (cases 1, 2, 3, and 4) presented with recurrent fever and splenomegaly, and two patients (cases 1 and 4) also suffered from rashes, hepatomegaly, and lymphadenectasis. Three patients (cases 1, 3, and 4) had thrombopenia, with or without leukopenia or erythropenia. Patient 1 had a history of agranulocytosis. Patient 3 previously worked in mountain fields, and therefore, insect bites were possible. Patient 5 was receiving chemotherapy. No predispos‐ ing factors were identified in cases 2 and 4. Bone marrow smears and cultures were performed for all five patients before referral to our hospital. Microorganisms were observed in bone marrow smears in cases 1, 2, and 5, but no definitive diagnosis by microscopic fea‐ tures was made. Only in case 2 was the initial clinical diagnosis by bone marrow smear later validated. In all five patients, symptoms did not improve after initial treatment at the local hospital; therefore, they were admitted to our hospital for further investigation. The baseline characteristics of the patients are listed in Table 1. All patients other than patient 2 provided consent for a second bone marrow aspiration, and metagenomics sequencing was performed using synchronously collected bone marrow and blood samples. For patient 2, only peripheral blood samples were collected and tested. Microorganisms were observed in bone marrow smears in cases 1 and 5 but not in cases 3 and 4 (Figure 1). Causative pathogens were detected by mNGS in all acquired bone marrow and blood samples for each case (Table 1), indicating the overall satisfactory pathogen detection ability of this approach. Leishmania infantum was detected in samples from cases 2, 3, and 5, and Histoplasma capsulatum and T marneffei were identified in cases 1 and 4, respectively. Rk39 se‐ rologic tests returned positive, confirming the diagnosis of visceral leishmaniasis. Bone marrow cultures were positive 14 days later in cases 1 and 4. After pathogen-specific treatment, the symptoms gradually im‐ proved in all patients. Serial bone marrow and blood samples from patients 1, 2, and 3 were also collected for mNGS according to clin‐ ical requirements. During follow-up visits, the sequencing reads in both bone marrow and peripheral blood samples obtained by mNGS were negative in cases 2 and 3 and showed reductions in read counts for pathogens in case 1, consistent with the improvements in clinical manifestations (Figure 2). Talaromycosis, histoplasmosis, and visceral leishmaniasis are po‐ tentially severe infections in humans and are commonly observed in immunocompromised patients or those living in epidemic areas. However, in China, owing to increases in population mobility, the in‐ cidence of AIDS, and the use of immunosuppressive agents, the ep‐ idemiological characteristics have become less typical. In our study, only two patients had typical susceptibility factors. The early diagnosis of these diseases is crucial in clinical set‐ tings because delays are associated with ineffective initial ther‐ apy and deterioration. However, diagnosis can be challenging owing to the similarities in clinical features (including fever, sple‐ nomegaly, or rash) and microscopic bone marrow morphology. Though Wright-Giemsa and periodic acid Schiff staining of bone marrow smears provide quick and robust evidence for early diag‐ nosis, the accuracy of these methods depends on the experience of pathologists.2,3 For 4 of 5 patients in our study, the initial clin‐ ical diagnosis by direct bone marrow smears and staining was in‐ accurate. This can possibly be explained by the fact that the four patients all received medical care in a nonepidemic area, where physicians might be less experienced in identifying the micro‐ organisms in bone marrow smears by microscopy. Additionally, Hao-Cheng Zhang, Qi-Ran Zhang, Jing-Wen Ai contributed equally to this manuscript.

The role of bone marrow metagenomics next-generation sequencing to differential diagnosis among visceral leishmaniasis, histoplasmosis, and talaromycosis marneffei.

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

The role of bone marrow metagenomics next-generation sequencing to differential diagnosis among visceral leishmaniasis, histoplasmosis, and talaromycosis marneffei.