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.

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CRISPR-based diagnostics.

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities

Growth in production of manufactured goods and the use of nanomaterials in consumer products has mounted in the past few decades. Nanotoxicology or toxicity assessment of these engineered products is required to understand possible adverse effects and their fate inside the human body. The present review is a one stop assessment intended to be a state of the art understanding on nanotoxicity. It provides a summation of the various kinds of cell death and also discusses the different types of toxicities along with their studies. The review discusses the physiological impact imparted on cells (reactive oxygen species generation and the resultant oxidative stress, inflammation, and other nonoxidant pathways). Moreover, it discusses the different physicochemical properties of nanomaterials (size, morphology, surface charge, and coating) governing the cytotoxicity properties. It also details the major pathways of nanomaterial uptake in cells and their outcome. Additionally, it also discusses the possible method...

Toxicity of Nanomaterials: Exposure, Pathways, Assessment, and Recent Advances

As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as “nanotransducers” that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light–activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.

Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.

A biodegradable two-dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered with three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3-BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation-associated endosomal escape and cytosolic releases of the loaded Cas9N3 complexes. The Cas9N3-BPs thus provide efficient genome editing and gene silencing in vitro and in vivo at a relatively low dose as compared with other nanoparticle-based delivery platforms. This biodegradable 2D delivery platform offers a versatile cytosolic delivery approach for CRISPR/Cas9 ribonucleoprotein and other bioactive macromolecules for biomedical applications.

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Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing.

Accurate and large-scale screening of infected individuals has proven to be an effective means to control the spread of COVID-19. Currently, many assays have been developed to meet the huge testing requirements and the availability of diverse detection settings. However, few methods emphasize the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Herein, we developed a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (ORF1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 µL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab clinical samples showed 100% negative predictive agreement (NPA) and 97.14% positive predictive agreement (PPA). Expectantly, this developed platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ange.202014506

Simultaneous dual-gene diagnosis of SARS-CoV-2 based on CRISPR/Cas9-mediated lateral flow assay.

The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called 'APC-Cas') that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 105 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.

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Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.

Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing and other recently developed applications, such as gene expression regulation and RNA/DNA imaging. Here, we first constructed a graphene oxide (GO)-polyethylene glycol (PEG)-polyethylenimine (PEI) nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes for endocytosis, endosomal escape, nuclear entry, and gene editing. The results demonstrate that the nanocarrier can be used successfully for efficient gene editing in human AGS cells with an efficiency of ∼39%. The results also show that this nanocarrier can protect sgRNA from enzymatic degradation, thus exhibiting extremely high stability, which is critical for future in vivo applications. Thus, this GO-mediated Cas9/sgRNA delivery system has potential as a new approach for biomedical research and targeted gene engineering applications.

Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.

Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.

Clustered, regularly interspaced short palindromic repeats (CRISPR)-based diagnoses, derived from gene-editing technology, have been exploited for less than 5 years and are now reaching the stage of precommercial use. CRISPR tools have some notable features, such as recognition at physiological temperature, excellent specificity, and high-efficiency signal amplification capabilities. These characteristics are promising for the development of next-generation diagnostic technologies. In this Perspective, we present a detailed summary of which micro/nanotechnologies play roles in the advancement of CRISPR diagnosis and how they are involved. The use of nanoprobes, nanochips, and nanodevices, microfluidic technology, lateral flow strips, etc. in CRISPR detection systems has led to new opportunities for CRISPR-based diagnosis assay development, such as achieving equipment-free detection, providing more compact detection systems, and improving sensitivity and quantitative capabilities. Although tremendous progress has been made, CRISPR diagnosis has not yet reached its full potential. We discuss upcoming opportunities and improvements and how micro/nanotechnologies will continue to play key roles.

Huahua Yue

Papers

Simultaneous dual-gene diagnosis of SARS-CoV-2 based on CRISPR/Cas9-mediated lateral flow assay.

Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.

Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing

Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies.