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Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms. CRISPR/Cas biosensing systems transfer the sequence information of target nucleic acids to detectable signals such as fluorescence and colorimetric values. CRISPR/Cas biosensing systems are versatile platforms for nucleic acid detection that can be used for pathogen detection and genotyping, cancer mutation detection, and single nucleotide polymorphism (SNP) identification. The biosensing methods employing these Cas effectors rely on the collateral cleavage activities of Cas13 and Cas12. CRISPR/Cas biosensing allows highly sensitive, specific, rapid, cost-efficient, and multiplex detection of target nucleic acids, and support point-of-care use without the need for technical expertise and complicated equipment.

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co‐infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis‐related factors (i.e., SPCSV‐RNase3) by using the CRISPR‐Cas13 technique. First, the RNA targeting activity of four CRISPR‐Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV‐RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3‐targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV‐GFP and cucumber mosaic virus CMV‐RNase3 co‐infection. Moreover, transgenic sweet potato plants carrying an RNase3‐targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD‐prevalent regions. Targeting of SPCSV‐RNase3 using CRISPR‐Cas13 (RfxCas13d) improves resistance against the sweet potato virus disease (SPVD) in transgenic sweet potato plants.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR–Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR–Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.

Background CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana . Results Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta . Conclusions Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) nucleases has transformed biotechnology by providing an easy, efficient, and versatile platform for editing DNA. However, traditional CRISPR-based technologies initiate editing by activating DNA double-strand break (DSB) repair pathways, which can cause adverse effects in cells and restrict certain therapeutic applications of the technology. To this end, several new CRISPR-based modalities have been developed that are capable of catalyzing editing without the requirement for a DSB. Here, we review three of these technologies: base editors, prime editors, and RNA-targeting CRISPR-associated protein (Cas)13 effectors. We discuss their strengths compared to traditional gene-modifying systems, we highlight their emerging therapeutic applications, and we examine challenges facing their safe and effective clinical implementation. Next-generation CRISPR technologies hold the potential to expand the capabilities of therapeutic editing.Advanced CRISPR modalities harness the versatility and programmability of traditional CRISPR systems but can catalyze highly precise editing outcomes without a DNA double‐strand break.Next-generation CRISPR technologies include base editors, prime editors, and RNA-targeting Cas13 effectors.The continued refinement of these technologies could enable their safe and effective implementation to treat a range of disorders.

We developed a sensitive and specific method based on recombinase‐aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated protein 13a (Cas13a). This method, named CRISPR‐based Rapid and Efficient Test (CRISPRET), is designed for the early diagnosis of Influenza B (FluB) with the aim of shortening its transmission chain. We identified conserved regions in the Influenza B Virus (IBV) NS gene and designed forward and reverse primers along with crRNAs. We then established and optimised the reaction system, and Nucleic Acid Positive Reference Materials of IBV were used to evaluate the detection limit (DL) of CRISPRET. Additionally, we collected 257 clinical samples, comprising 127 samples from patients with IBV infection and 130 samples from healthy individuals, and subjected them to dual detection using CRISPRET and qPCR to evaluate the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of CRISPRET. We designed one forward primer, two reverse primers, and two crRNAs to establish and optimise the CRISPR ET. The method demonstrated the DL of 500 copies·μL−1 when assisted by appropriate equipment. Despite requiring auxiliary equipment and a 30‐min reaction, the CRISPR ET method enables the detection of IBV nucleic acid within approximately the first 5 min, achieving high sensitivity (100%), specificity (97.69%), PPV (97.69%) and NPV (100%), with a concordance rate of 98.83% to qPCR. CRISPRET offers a simple, field‐applicable, one‐step method for the rapid detection of IBV. It has strong potential for field‐testing applications and intelligent integration into existing diagnostic systems. CRISPRET initially designs upstream and downstream primers and crRNAs targeting the NS gene of the Influenza B Virus (FluB) for the identification of the target RNA (NS gene). During testing, RNA from the Influenza B Virus is extracted from clinical throat swab samples using a sample RNA release preservative. The obtained RNA is then combined with the CRISPR‐Cas13a‐targeted RNA system, which includes an RNA fluorescent probe labelled with FAM and BHQ. The samples to be tested were placed into the test wells of the Genchek Fluorometer at a constant temperature of 37°C. The instrument automatically collects fluorescence every 20 s, and the entire reaction curve was obtained after 30 min of reaction. By comparing the results from the Genchek fluorimeter and qPCR, CRISPRET demonstrates high sensitivity (100%), specificity (97.69%), positive predictive value (97.69%), negative predictive value (100%), and an overall concordance rate of 98.83%.

Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila , both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

Monitoring clinically relevant antibodies—as biomarkers of disease or therapeutic response—is essential for informed clinical decision‐making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point‐of‐care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR‐Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity‐triggered Linker Exchange), a modular CRISPR‐Cas13–based platform for ultrasensitive antibody detection. MARPLE harnesses antibody‐induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13‐mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets—including anti‐digoxigenin, anti‐cholesterol, anti‐HA, trastuzumab, and anti‐MUC1—highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one‐pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR‐based sensing beyond nucleic acids to protein biomarker detection. MARPLE is a modular CRISPR‐Cas13 immunoassay that uses antibody‐induced proximity to trigger a toehold‐exchange reaction, releasing an RNA activator for Cas13. This one‐pot, isothermal architecture enables ultrasensitive (femtomolar) and highly specific detection of diverse antibodies, including therapeutic, pathogen‐specific, and cancer‐associated targets, with robust performance in human serum.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology can detect both nucleic acid and non-nucleic acid (NNA) targets.CRISPR-based NNA detection systems use CRISPR-associated (Cas) trans-cleavage as a signal amplifier.The specificity of CRISPR-based NNA detection systems is determined by NNA-targeting strategies, including aptamers, DNAzymes, allosteric transcription factors (aTFs), antibodies, and cofactor-based biochemical reactions.The underlying mechanism of CRISPR-based NNA detection is to link the presence of a target NNA to the accessibility of three essential CRISPR components: the activator, CRISPR RNA (crRNA), and Cas effector.CRISPR-based NNA detection is sensitive, rapid, and free of aerosol contamination risk, and can be used in both clinical and nonclinical scenarios. Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection. Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.