Rapid and sensitive detection of specific RNA sequences is crucial for clinical diagnosis, surveillance, and biotechnology applications. Currently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard for RNA detection; however, it is associated with long processing time, complex procedures, and a high false-positive rate. To address these challenges, we developed a novel sensing platform based on CRISPR/Cas13a that incorporates a dumbbell-shaped hairpin and DNA-PAINT for rapid, highly specific, and sensitive RNA analysis. By leveraging the CRISPR/Cas13a system, this platform enables the cleavage of dumbbell-shaped hairpins, which subsequently allows the cleaved primers to initiate cyclic amplification of fluorescent signals. These signals are further enhanced by the binding and dissociation phenomena inherent to DNA-PAINT technology, ultimately achieving remarkable triple signal amplification. Additionally, the system effectively discriminates Hantaan virus RNA from Seoul virus in real samples. Importantly, the platform can be easily adapted for the detection of other RNAs by simply reconfiguring the hybridization region of crRNA. In conclusion, this platform represents a "five-in-one" RNA detection approach that integrates reliability, versatility, robustness, high specificity, and superior quantitative capabilities. It provides novel insights for direct RNA detection based on CRISPR/Cas13a and demonstrates significant potential for advancement in viral diagnostics.

RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.

Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA. We show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. Early phage genes are expressed from the EPI vesicle, demonstrating its functionality as a prokaryotic, transcriptionally active, membrane-bound organelle. We also show that the phage nucleus is essential, with genome replication beginning after the injected DNA is transferred from the EPI vesicle to the phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

Recent advancements in molecular engineering have established RNA-based technologies as powerful tools for both fundamental research and translational applications. Among the various RNA-based technologies developed, RNA base editing has recently emerged as a groundbreaking advancement. It primarily involves the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which are mediated by ADAR and APOBEC enzymes, respectively. RNA base editing has been applied in both biological research and therapeutic contexts. It enables site-directed editing within target transcripts, offering reversible, dose-dependent effects, in contrast to the permanent or heritable changes associated with DNA base editing. Additionally, RNA editing-based profiling of RNA-binding protein (RBP) binding sites facilitates transcriptome-wide mapping of RBP-RNA interactions in specific tissues and at the single-cell level. Furthermore, RNA editing-based sensors have been utilized to express effector proteins in response to specific RNA species. As RNA base editing technologies continue to evolve, we anticipate that they will significantly drive advancements in RNA therapeutics, synthetic biology, and biological research.

Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.

Type V and type VI CRISPR-Cas systems have been shown to cleave nonspecific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) in trans, but this has not been observed in type II CRISPR-Cas systems using single guide RNA. We show here that the type II CRISPR-Cas9 systems directed by CRISPR RNA and trans-activating CRISPR RNA dual RNAs show RuvC domain-dependent trans-cleavage activity for both ssDNA and ssRNA substrates. Cas9 possesses sequence preferences for trans-cleavage substrates, preferring to cleave T- or C-rich ssDNA substrates. We find that the trans-cleavage activity of Cas9 can be activated by target ssDNA, double-stranded DNA and ssRNA. The crystal structure of Cas9 in complex with guide RNA and target RNA provides a structural basis for the binding of target RNA to activate Cas9. Based on the trans-cleavage activity of Cas9 and nucleic acid amplification technology, we develop the nucleic acid detection platforms DNA-activated Cas9 detection and RNA-activated Cas9 detection, which are capable of detecting DNA and RNA samples with high sensitivity and specificity.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.

The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.

Plant viruses pose a significant threat to global agriculture. For a long time, conventional methods including detection based on visual symptoms, host range investigations, electron microscopy, serological assays (e.g., ELISA, Western blotting), and nucleic acid-based techniques (PCR, RT-PCR) have been used for virus identification. With increased sensitivity, speed, and specificity, new technologies like loop-mediated isothermal amplification (LAMP), high-throughput sequencing (HTS), nanotechnology-based biosensors, and CRISPR diagnostics have completely changed the way plant viruses are detected. Recent advances in detection techniques integrate artificial intelligence (AI), machine learning (ML), and the Internet of Things (IoT) for real-time monitoring. Innovations like hyperspectral imaging, deep learning, and cloud-based IoT platforms further support disease identification and surveillance. Nanotechnology-based lateral flow assays and CRISPR-Cas systems provide rapid, field-deployable solutions. Despite these advancements, challenges such as sequence limitations, multiplexing constraints, and environmental concerns remain. Future research should focus on refining portable on-site diagnostic kits, optimizing nanotechnology applications, and enhancing global surveillance systems. Interdisciplinary collaboration across molecular biology, bioinformatics, and engineering is essential to developing scalable, cost-effective solutions for plant virus detection, ensuring agricultural sustainability and ecosystem protection.

CRISPR-Cas systems represent a highly programmable and precise nucleic acid-targeting platform, which has been strategically engineered as a versatile toolkit for biosensing and bioimaging applications. Nevertheless, their analytical performance is constrained by inherent functional and activity limitations of natural CRISPR/Cas systems, underscoring the critical role of molecular engineering in enhancing their capabilities. This review comprehensively examines recent advancements in engineering CRISPR/Cas ribonucleoproteins (RNPs) to enhance their functional capabilities for advanced molecular detection and cellular imaging. We explore innovative strategies for developing enhanced CRISPR/Cas RNPs, including Cas protein engineering through protein mutagenesis and fusion techniques, and guide RNA engineering via chemical and structural modifications. Furthermore, we evaluate these engineered RNPs' applications in sensitive biomarker detection and live-cell genomic DNA and RNA monitoring, while analyzing the current challenges and prospective developments in CRISPR-Cas RNP engineering for advanced biosensing and bioimaging.

Circular RNAs (circRNAs) constitute an RNA type formed by back-splicing. BCL2-like 12 (BCL2L12) is an apoptosis-related gene comprising 7 exons. In this study, we used targeted nanopore sequencing to identify circular BCL2L12 transcripts in human colorectal cancer cells and investigated the effect of circRNA silencing on mRNA expression of the parental gene. In brief, nanopore sequencing following nested PCR amplification of cDNAs of BCL2L12 circRNAs from 7 colorectal cancer cell lines unraveled 46 BCL2L12 circRNAs, most of which described for the first time. Interestingly, 40 novel circRNAs are likely to form via back-splicing between non-canonical back-splice sites residing in highly similar regions of the primary transcripts. All back-splice junctions were validated using next-generation sequencing (NGS) after circRNA enrichment. Surprisingly, 2 novel circRNAs also comprised a poly(A) tract after BCL2L12 exon 7; this poly(A) tract was back-spliced to exon 1, in both cases. Furthermore, the selective silencing of a BCL2L12 circRNA resulted in a subsequent decrease of BCL2L12 mRNA levels in HCT 116 cells, thus providing evidence of parental gene expression regulation by circRNAs. In conclusion, our study led to the discovery of many circular transcripts from a single human gene and provided new insights into circRNA biogenesis and mode of action.

CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.

Simultaneous targeting of multiple loci with the CRISPR system, a tool known as multiplex CRISPR, offers greater feasibility for manipulating and elucidating the intricate and redundant endogenous networks underlying complex cellular functions. Owing to the versatility of continuously emerging Cas nucleases and the use of CRISPR arrays, multiplex CRISPR has been implemented in numerous in vitro and in vivo studies. However, a streamlined, practical strategy for CRISPR array assembly that is both convenient and accurate is lacking. Here, we present a novel, highly accurate, cost-, and time-saving strategy for CRISPR array assembly. Using this strategy, we efficiently assembled 12 CRISPR RNAs (crRNAs) (for AsCas12a) and 15 crRNAs (for RfxCas13d) in a single reaction. CRISPR arrays driven by Pol II promoters exhibited a distinct expression pattern compared with those driven by Pol III promoters, which could be exploited for specific distributions of CRISPR intensity. Improved approaches were subsequently designed and validated for expressing long CRISPR arrays. The study provides a flexible and powerful tool for the convenient implementation of multiplex CRISPR across DNA and RNA, facilitating the dissection of sophisticated cellular networks and the future realization of multi-target gene therapy.

Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (105 to 102 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.

Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Control of Trypanosoma brucei evansi (T. b. evansi) infections remains a significant challenge in managing Surra, a widespread veterinary disease affecting both wild and domestic animals. In the absence of an effective vaccine, accurate diagnosis followed by treatment is crucial for successful disease management. However, existing diagnostic methods often fail to detect active infections, particularly in field conditions. Recent advancements in CRISPR-Cas technology, combined with state-of-the-art isothermal amplification assays, offer a promising solution. This approach has led us to the development of a TevRPA-CRISPR assay, a highly sensitive and specific T. b. evansi diagnostic tool suitable for both laboratory and field settings.

First, the TevCRISPR-Cas12b cleavage assay was developed and optimized, and its analytical sensitivity was evaluated. Next, this technology was integrated with the TevRPA to create the TevRPA-CRISPR test, with the reaction conditions being optimized and its analytical sensitivity and specificity assessed. Finally, the test's accuracy in detecting both active and cured T. b. evansi infections was evaluated.

The optimized TevCRISPR-Cas12b cleavage assay demonstrated the ability to detect T. b. evansi target DNA at picomolar concentrations. Integrating TevCRISPR-Cas12b with RPA in Two-Pot and One-Pot TevRPA-CRISPR tests achieved up to a 100-fold increase in analytical sensitivity over RPA alone, detecting attomolar concentrations of T. b. evansi target DNA, while maintaining analytical specificity for T. b. evansi. Both assays exhibited performance comparable to the gold standard TevPCR in experimental mouse infections, validating their effectiveness for detecting active infections and assessing treatment efficacy.

The TevRPA-CRISPR tests prove highly effective for diagnosing active infections and assessing treatment efficacy, while being adaptable for both laboratory and field use. Thus, the TevRPA-CRISPR assays emerge as a promising addition to current diagnostic tools, offering efficient and reliable detection of active T. b. evansi infections.

CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare the Cas13 effectors PspCas13b and Cas13bt3 for the repair of the gene USH2A, a common cause of inherited retinal disease and Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency for the repair of the common c.11864 G > A and its murine equivalent c.11840 G > A, across different gRNAs and promoters. We develop and characterize a mouse model of Usher syndrome carrying the c.11840 G > A mutation designed for the evaluation of base editors for inherited retinal disease. Finally, we compare Cas13 effectors delivered via AAV for the repair of Ush2a in photoreceptors. Mean RNA editing rates in photoreceptors across different constructs ranged from 0.32% to 2.04%, with greater efficiency in those injected with PspCas13b compared to Cas13bt3 constructs. In mice injected with PspCas13b constructs, usherin protein was successfully restored and correctly localized to the connecting cilium following RNA editing. These results support the development of transcriptome targeting gene editing therapies for retinal disease.

Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.

Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .

Genome and transcriptome engineering have emerged as powerful tools in modern biotechnology, driving advancements in precision medicine and novel therapeutics. In this review, we provide a comprehensive overview of the current methodologies, applications, and future directions in genome and transcriptome engineering. Through this, we aim to provide a guide for tool selection, critically analyzing the strengths, weaknesses, and best use cases of these tools to provide context on their suitability for various applications. We explore standard and recent developments in genome engineering, such as base editors and prime editing, and provide insight into tool selection for change of function (knockout, deletion, insertion, substitution) and change of expression (repression, activation) contexts. Advancements in transcriptome engineering are also explored, focusing on established technologies like antisense oligonucleotides (ASOs) and RNA interference (RNAi), as well as recent developments such as CRISPR-Cas13 and adenosine deaminases acting on RNA (ADAR). This review offers a comparison of different approaches to achieve similar biological goals, and consideration of high-throughput applications that enable the probing of a variety of targets. This review elucidates the transformative impact of genome and transcriptome engineering on biological research and clinical applications that will pave the way for future innovations in the field.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.

HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.

The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.

CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.

These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.

We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

Circular RNA (circRNA) and microRNA (miRNA) are both non-coding RNAs (ncRNAs) that serve as biomarkers for cancer diagnosis and prognosis. Quantitative detection of these ncRNAs is of particular importance to elucidate the functional mechanisms and evaluate their potential as biomarkers. However, the inherent structures of circRNA and miRNA are different from the mRNA, conventional qRT-PCR is unsuitable for the detection of these ncRNAs. Here, we propose a sensitive method for quantitative detection of circRNA and miRNA using polydisperse droplet digital CRISPR/Cas13a (PddCas13a). It can achieve limits of detection (LOD) as low as ∼10 aM without polymerase-based amplification. To efficiently detect the circRNA and miRNA in real samples, we use a chemically modified crRNA to enhance the stability of crRNA and improve the performance of Cas13a in complex environments containing contaminants. By integrating an extraction-free procedure with PddCas13a, we experimentally demonstrate the applicability of PddCas13a by testing clinical samples. Furthermore, we develop an automated and portable instrument for PddCas13a and verify its applicability for the detection of circRNA and miRNA from exosomes in point-of-care (POC) setting. This is the first report to detect the circRNA and miRNA simultaneously in POC setting. We envision this platform could promote the research of ncRNAs.

Creating fast, non-invasive, precise, and specific diagnostic tests is crucial for enhancing cancer treatment outcomes. Among diagnostic methods, those relying on nucleic acid detection are highly sensitive and specific. Recent developments in diagnostic technologies, particularly those leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are revolutionizing cancer detection, providing accurate and timely results. In clinical oncology, liquid biopsy has become a noninvasive and early-detectable alternative to traditional biopsies over the last two decades. Analyzing the nucleic acid content of liquid biopsy samples, which include Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), Circulating Cell-Free RNA (cfRNA), and tumor extracellular vesicles, provides a noninvasive method for cancer detection and monitoring. In this review, we explore how the characteristics of various Cas (CRISPR-associated) enzymes have been utilized in diagnostic assays for cancer liquid biopsy and highlight their main applications of innovative approaches in monitoring, as well as early and rapid detection of cancers.

RNAs are increasingly recognized as promising therapeutic targets, susceptible to modulation by strategies that include targeting with small molecules, antisense oligonucleotides, deoxyribozymes (DNAzymes), or CRISPR/Cas13. However, while drug development for proteins follows well-established paths for rational design based on the accurate knowledge of their three-dimensional structure, RNA-targeting strategies are challenging since comprehensive RNA structures are yet scarce and challenging to acquire. Numerous methods have been developed to elucidate the secondary and three-dimensional structure of RNAs, including X-ray crystallography, cryo-electron microscopy, nuclear magnetic resonance, SHAPE, DMS, and bioinformatic methods, yet they have often revealed flexible transcripts and co-existing populations rather than single-defined structures. Thus, researchers aiming to target RNAs face a critical decision: whether to acquire the detailed structure of transcripts in advance or to adopt phenotypic screens or sequence-based approaches that are independent of the structure. Still, even in strategies that seem to rely only on the nucleotide sequence (like the design of antisense oligonucleotides), researchers may need information about the accessibility of the compounds to the folded RNA molecule. In this concise guide, we provide an overview for researchers interested in targeting RNAs: We start by revisiting current methodologies for defining secondary or three-dimensional RNA structure and then we explore RNA-targeting strategies that may or may not require an in-depth knowledge of RNA structure. We envision that complementary approaches may expedite the development of RNA-targeting molecules to combat disease.

Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.

Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host-pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs, and it complements existing amplification-based approaches.

Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.

CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, is currently employed for RNA degradation. However, the potential of various CRISPR/Cas13 subtypes, particularly concerning the knockdown of endogenous transcripts, remains to be comprehensively characterized in plants.

Here we present a full spectrum of editing profiles for seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 and Cas13x.2), and VI-Y (Cas13y.1 and Cas13y.2). A systematic evaluation of the knockdown effects on two endogenous transcripts (GhCLA and GhPGF in cotton) as well as an RNA virus (TMV in tobacco) reveals that RfxCas13d, Cas13x.1, and Cas13x.2 exhibit enhanced stability with editing efficiencies ranging from 58 to 80%, closely followed by Cas13y.1 and Cas13y.2. Notably, both Cas13x.1 and Cas13y.1 can simultaneously degrade two endogenous transcripts through a tRNA-crRNA cassette approach, achieving editing efficiencies of up to 50%. Furthermore, different Cas13 orthologs enable varying degrees of endogenous transcript knockdown with minimal off-target effects, generating germplasms that exhibit a diverse spectrum of mutant phenotypes. Transgenic tobacco plants show significant reductions in damage, along with mild oxidative stress and minimal accumulation of viral particles after TMV infection.

In conclusion, our study presents an efficient and reliable platform for transcriptome editing that holds promise for plant functional research and future crop improvement.

Histone citrullination, an important post-translational modification mediated by peptidyl arginine deiminases, is essential for many physiological processes and epigenetic regulation. However, the causal relationship between histone citrullination and specific gene regulation remains unresolved. In this study, we develop a programmable epigenetic editor by fusing the peptidyl arginine deiminase (PAD) PPAD from Porphyromonas gingivalis with dCas9. With the assistance of gRNA, PPAD-dCas9 can recruit PPADs to specific genomic loci, enabling direct manipulation of the epigenetic landscape and regulation of gene expression. Our citrullination editor allows for the site-specific manipulation of histone H3R2,8,17 and H3R26 at target human gene loci, resulting in the activation or suppression of different genes in a locus-specific manner. Moreover, the epigenetic effects of the citrullination editor are specific and sustained. This epigenetic editor offers an accurate and efficient tool for exploring gene regulation of histone citrullination.