Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per µL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

CRISPR­based diagnostic approaches: Implications for rapid management of future pandemics (Review).

Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SARS­CoV), Middle East respiratory syndrome corona virus, and SARS­CoV­2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human­associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in China and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold­standard molecular diagnostic approaches currently used are time­consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point­of­care devices for widespread monitoring and surveillance. Clustered regularly interspaced short palindromic repeats (CRISPR)­associated (Cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. CRISPR­Cas systems are organized into CRISPR arrays and adjacent Cas proteins. The detection and in­depth biochemical characterization of class 2 type V and VI CRISPR­Cas systems and orthologous proteins such as Cas12 and Cas13 have led to the development of CRISPR­based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. CRISPR­based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain RNA as a genome. CRISPR­based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of Cas12 and Cas13 orthologs in viral disease detection and other applications. The present review expands the scope of CRISPR­based diagnostic approaches to detect diseases and fight viruses as antivirals.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

FELICX: A robust nucleic acid detection method using flap endonuclease and CRISPR-Cas12.

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/µL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.

A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

An aM-level sensitive cascade CRISPR-Dx system (ASCas) for rapid detection of RNA without pre-amplification.

The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.

Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/µL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.

Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.

Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.

CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.

Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.

Nanoparticles-mediated CRISPR-Cas9 gene therapy in inherited retinal diseases: applications, challenges, and emerging opportunities.

Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.

[CRISPR-based molecular diagnostics: a review].

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking ß-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of ß-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.