CRISPR Research Articles

Transcriptional profiles of Microcystis reveal gene expression shifts that promote bloom persistence in in situ mesocosms

ABSTRACT Harmful algal blooms caused by cyanobacteria threaten aquatic ecosystems, the economy, and human health. Previous work has tried to identify the mechanisms that allow blooms to form, focusing on the role of nutrients. However, little is known about how introduced nutrients influence gene expression in situ . To address this knowledge gap, we used in situ mesocosms initiated with water experiencing a Microcystis bloom. We added pulses of nutrients that are commonly associated with anthropogenic sources to the mesocosms for 72 hours and collected samples for metatranscriptomics to examine how the physiological function of Microcystis and bloom status changed. The addition of nitrogen (N) as urea, but not the addition of PO 4 , resulted in conspicuous bloom persistence for at least 9 days after the final introduction of nutrients. The addition of urea initially resulted in the upregulation of photosynthesis machinery, as well as phosphate, carbon, and N transport and metabolism. Once Microcystis presumably became N-replete, upregulation of amino acid metabolism, microcystin biosynthesis, and other processes associated with biomass generation occurred. These capacities coincided with the upregulation of toxin-antitoxin systems, CRISPR- cas genes, and transposases suggesting that phage defense and genome rearrangement are critical in bloom persistence. Overall, our results show the stepwise transcriptional response of a Microcystis bloom to the introduction of nutrients, specifically urea, as it is sustained in a natural setting. The transcriptomic shifts observed herein may serve as markers of the longevity of blooms while providing insight into why Microcystis blooms over other cyanobacteria. IMPORTANCE Harmful algal blooms represent a threat to human health and ecosystems. Understanding why blooms persist may help us develop warning indicators of bloom persistence and create novel mitigation strategies. Using mesocosm experiments initiated with water with an active bloom, we measured the stepwise transcription changes of the toxin-producing cyanobacterium Microcystis in response to the addition of nutrients that are important in causing blooms. We found that nitrogen (N), but not phosphorus, promoted bloom longevity. The initial introduction of N resulted in the upregulation of genes involved in photosynthesis and N import. At later times in the bloom, upregulation of genes involved in biomass generation, phage protection, genomic rearrangement, and toxin production was observed. Our results suggest that Microcystis first fulfills nutritional requirements before investing energy in pathways associated with growth and protection against competitors, which allowed bloom persistence more than a week after the final addition of nutrients.

Synthetic Biology-Enabled Engineering of Probiotics for Precision and Targeted Therapeutic Delivery Applications

Probiotics, defined as live microorganisms that offer health benefits when consumed in adequate amounts, have traditionally been used to maintain gastrointestinal (GI) health. They are effective in managing conditions such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and antibiotic-associated diarrhea. Recent research, however, has expanded their potential applications beyond the GI tract to include immune modulation, metabolic regulation, and mental health support. Despite these advances, conventional probiotics face limitations such as instability in harsh environments, lack of precise targeting, and ineffective control over drug release. This review explores the application of synthetic biology in addressing these limitations, emphasizing how tools such as CRISPR-Cas systems, synthetic gene circuits, and plasmid-based mechanisms enhance the stability, functionality, and specificity of engineered probiotics for therapeutic delivery. Applications of synthetic biology-enabled probiotics are examined across a range of health contexts, including GI health, cancer therapy, metabolic disorders, immune modulation, and mental well-being. Synthetic biology tools provide precise genetic modifications that improve the stability, viability, and functionality of engineered probiotics, enabling more targeted therapeutic delivery. Additionally, technologies such as encapsulation support probiotic survival, while gene circuits allow programmable, stimulus-responsive therapeutic release. As synthetic biology continues to advance, the development of programmable probiotics holds promise for personalized, targeted medicine, positioning probiotics as living therapeutics and patient-friendly alternatives to conventional therapeutic delivery systems.

Discovery and characterization of complete genomes of 38 head-tailed proviruses in four predominant phyla of archaea.

Archaea play a significant role in natural ecosystems and the human body. Archaeal viruses exert a considerable influence on the structure and composition of archaeal communities and their associated ecological environments. The present study revealed the complete genomes of 38 archaeal head-tailed proviruses through comprehensive data mining. The hosts of these proviruses were identified as belonging to the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. In addition to the 14 proviruses of halophilic archaea related to the Graaviviridae family, the remaining proviruses exhibited limited genetic similarities to known (pro)viruses, suggesting the existence of 14 potential novel families. Of the 38 archaeal proviruses, 30 have the potential to lyse host cells. Eleven proviruses contain genes linked to antiviral defense mechanisms, including those involved in restriction modification (RM), clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) nucleases, defense island system associated with restriction-modification (DISARM), and DNA degradation (Dnd). Moreover, auxiliary metabolic genes were identified in the proviruses of Bathyarchaeia and Halobacteriota archaea, including those involved in carbohydrate and amino acid metabolism. Our findings indicate the diversity of archaeal viruses, their interactions with archaeal hosts, and their roles in the adaptation of the host.IMPORTANCEThe field of archaeal virology has seen a rapid expansion through the use of metagenomics, yet the diversity of these viruses remains largely uncharted. In this study, the complete genomes of 38 novel archaeal proviruses were identified for the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. Two families and six genera of Archaea were the first to be identified as hosts for viruses. The proviruses were found to contain diverse genes that were involved in distinct adaptation strategies of viruses to hosts. Our findings contribute to the expansion of the lineages of archaeal viruses and highlight their intricate interactions and essential roles in enabling host survival and adaptation to diverse environmental conditions.

Development of an optimized protocol for generating knockout cancer cell lines using the CRISPR/Cas9 system, with emphasis on transient transfection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system offers cost-effectiveness, high efficiency, precision, and ease of use compared to traditional gene editing techniques. In this study, we employed findings from prestigious investigations to develop an optimized approach for generating knockout cancer cell lines using a transient transfection method. This protocol introduces a distinctive approach that follows rigorous guidelines for designing gRNA to reduce off-target effects, a major challenge in CRISPR applications. Our step-by-step instructions allow researchers, particularly those with limited laboratory equipment and funding, as well as those undertaking CRISPR projects for the first time, to generate knockout cell lines using CRISPR technology in just ten weeks. This protocol covers all needs for enhancing various yields, such as transfection efficiency, and includes leveraging robust bioinformatics tools, conducting essential assays, isolating monoclonal cells via limiting dilution, validating knockout cells, and providing comprehensive troubleshooting recommendations. Using this method, we successfully created several new generations of colorectal cancer cell lines with monoallelic and biallelic knockouts of the epithelial cell adhesion molecule (EpCAM) gene. Our method, optimized for a wide spectrum of cancer cell lines, makes CRISPR more accessible for applications in personalized and precision medicine. It expands opportunities for novel investigations into cancer mechanisms and paves the way for potential therapeutic interventions.

Endometriosis: Future Biological Perspectives for Diagnosis and Treatment

Endometriosis is an oestrogen-dependent inflammatory disease affecting menstruating women, with varying levels of severity. Oestrogen dysregulation is responsible for chronic inflammation, angiogenesis, endometrial lesion development, progression, and infertility during menarche in afflicted women. The inflammatory mediators associated with this chronic painful disease have been established, with research also indicating the relationship between dysbiosis and disease manifestation. Endometriosis is also present with several painful comorbidities, including endometrial cancer, cardiovascular disease, and autoimmunity. The lack of specific and sensitive non-invasive diagnostic procedures, coupled with poor response to current therapeutic approaches, means that treatment needs remain unmet. Surgical procedures are performed to remove endometriosis ectopic lesions, for which the recurrence rate of disease is up to 50%, with certain patients exhibiting no alleviation of symptoms. This review aims to outline the aetiology of endometriosis, detailing novel diagnostic approaches and potential therapeutic approaches, namely advanced therapeutic medical products (ATMPs), including stem cell therapy and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing. This timely review also provides novel insights into the important recent modalities which may be applied for the diagnosis and therapeutic response of endometriosis, including biomarkers, microfluidic platforms, and organoid systems. Undoubtedly, reliable, reproducible, sensitive, and specific models of endometriosis in humans are urgently needed to investigate and detail the aetiology of this debilitating disease.

Genetic Engineering in Bacteria, Fungi, and Oomycetes, Taking Advantage of CRISPR

Genetic engineering has revolutionized our ability to modify microorganisms for various applications in agriculture, medicine, and industry. This review examines recent advances in genetic engineering techniques for bacteria, fungi, and oomycetes, with a focus on CRISPR-Cas systems. In bacteria, CRISPR-Cas9 has enabled precise genome editing, enhancing applications in antibiotic production and metabolic engineering. For fungi, despite challenges associated with their complex cell structures, CRISPR/Cas9 has advanced the production of enzymes and secondary metabolites. In oomycetes, significant plant pathogens, modified Agrobacterium-mediated transformation, and CRISPR/Cas12a have contributed to developing disease-resistant crops. This review provides a comparative analysis of genetic engineering efficiencies across these microorganisms and addresses ethical and regulatory considerations. Future research directions include refining genetic tools to improve efficiency and expand applicability in non-model organisms. This comprehensive overview highlights the transformative potential of genetic engineering in microbiology and its implications for addressing global challenges in agriculture, medicine, and biotechnology.

A Novel High-Throughput Sample-in-Result-Out Device for the Rapid Detection of Viral Nucleic Acids

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

CRISPR-Cas13a-Based Lateral Flow Assay for Detection of Bovine Leukemia Virus

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), which presents worldwide prevalence. BLV caused substantial economic loss in China around the 1980s; then, it could not be detected for some time, until recently. Due to its latent and chronic characteristics, the efficient and accurate detection of BLV is of utmost significance to the timely implementation of control measures. Therefore, this study harnessed the recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) technology, and lateral flow (LF) strips to develop an efficient method for detection of BLV. In this method, isothermal amplification of the targeted pol gene is performed at 37 °C with a detection threshold of 1 copy/µL, and the procedure is completed in 100 min. This assay demonstrated high selectivity for BLV, as indicated by the absence of a cross-reaction with six common bovine pathogens. Remarkably, 100 blood samples from dairy cows were tested in parallel with a conventional quantitative polymerase chain reaction (qPCR) and this method and the results showed 100% agreement. Furthermore, this method exhibited good repeatability. In conclusion, in this study, we established a sensitive and specific method for BLV detection, which shows promise for application in BLV surveillance.

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

End-point diagnostics of Giardia duodenalis assemblages A and B by combining RPA with CRISPR/Cas12a from human fecal samples.

Giardia duodenalis is a common enteric protozoan parasite that is categorized into eight assemblages (A-H). In particular, assemblages A and B are zoonotic, capable of infecting both humans and animals worldwide, resulting in significant economic losses and public health challenges in epidemic regions. Thus, the development of rapid, accurate and non-laboratory-based diagnostic methods for infected animals is crucial for the effective prevention and control of giardiasis. Recent advancements in clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein (Cas12a) systems allow promising avenues for nucleic acid detection, characterized by their high flexibility, sensitivity and specificity. Combined recombinase polymerase amplification and CRISPR/Cas12a systems were combined and used as end-point diagnostic methods (termed REPORT) to detect G. duodenalis assemblage A and B. The diagnostic results can be observed by fluorescence readouts with the naked eye under blue light or colorimetric signals using a lateral flow strip (LFS). The limit of detection (LOD) of the REPORT‑based G. duodenalis assemblage A detection was 2.04 CFU/ml and 10 trophozoites per gram (TPG), and the LOD of assemblage B was 1.1 CFU/ml and 10 cysts per gram (CPG). The REPORT‑based G. duodenalis assemblage A and assemblage B detection methods have strong specificity and no cross-reactivity with other assemblages of G. duodenalis or common enteric parasitic protozoa and have excellent performance in clinical sample detection. This study presents a novel strategy for the direct identification of G. duodenalis assemblages A and B, requiring neither highly trained personnel nor costly specialized equipment.

Proliferation Analyses of Conditional Knockdown Strains Using CRISPR Interference in Fission Yeast.

CRISPR interference is a method to conditionally inhibit transcription of an arbitrary target gene. This is useful to study the functions of essential genes, which are required for cellular viability. Although many conditional gene perturbation techniques are available for Schizosaccharomyces pombe, CRISPRi facilitates construction of a large number of knockdown strains because of its systematic, simple procedure. Here, we describe a method to construct and characterize knockdown strains using dCas9-mediated CRISPRi in S. pombe, including a variation of CRISPRi induction technique in a 96-well format for high-throughput studies.

Abstract 4116949: Specialized Pericyte Subtypes in the Pulmonary Capillary

Introduction: Lung pericytes (PCs) are mural cells in close contact with endothelial cells (ECs) in the microvasculature. A significant challenge in investigating PC biology is largely due to the absence of a unique cell marker, making it difficult to distinguish them from other mural cell populations. The identification of such a marker would allow investigators to describe the role of PCs in various diseases including pulmonary arterial hypertension. Hypothesis: We hypothesize that HIG1 hypoxia-inducible domain family member 1B ( Higd1b ) is exclusively expressed in lung PCs, which contributes to hypoxia (Hx)-induced vascular remodeling. Methods: We utilized single-cell RNA sequence (scRNA-seq) databases, spatial transcriptomics, and RNAscope from human and murine lungs to identify a PC-specific cell marker and compare the gene expression between PC subtypes. We utilized Cre-LoxP and CRISPR technology to construct a novel tamoxifen-inducible Higd1b-CreERT2 knock-in mouse model and performed lineage-tracing studies to describe the role of PC subtypes in Hx-induced pulmonary hypertension (PH). Results: ScRNA-seq analysis from the lungs of humans and mice identified Higd1b as a specific gene marker for PCs whose expression is absent in other mural cell populations. Validation with a reporter mouse line Higd1b-CreERT2::R26-tdT confirmed Higd1b-Cre+ cells specifically label PCs and no other mural cells ( Fig 1A ). Lineage tracing in the Hx-induced murine model of PH demonstrated the accumulation of PCs in the muscularized distal arterioles ( Fig 1B ). Through scRNA-seq and immunofluorescence validation, we identified two HIGD1B + PC subtypes that exist in the pulmonary capillary: Type 1 PCs, which are quiescent in the capillaries, and Type 2 PCs exhibit multipotent cell-like properties and accumulate in the arterioles after exposure to Hx. Furthermore, we found Type 2 PCs transited into SMC-like cells via the upregulation of Vimentin, contributing to vascular remodeling in Hx-induced PH. Conclusion: We identified Higd1b as a unique marker for PCs and generated a novel Higd1b-CreERT2 mouse that specifically labels PCs in the lungs. The discovery of PC-subtype specialization advances our understanding of lung pericyte biology and PC’s contribution to capillary remodeling under pathological conditions.

CRISPRoffT: comprehensive database of CRISPR/Cas off-targets.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) programmable nuclease system continues to evolve, with in vivo therapeutic gene editing increasingly applied in clinical settings. However, off-target effects remain a significant challenge, hindering its broader clinical application. To enhance the development of gene-editing therapies and the accuracy of prediction algorithms, we developed CRISPRoffT (https://ccsm.uth.edu/CRISPRoffT/). Users can access a comprehensive repository of off-target regions predicted and validated by a diverse range of technologies across various cell lines, Cas enzyme variants, engineered sgRNAs (single guide RNAs) and CRISPR editing systems. CRISPRoffT integrates results of off-target analysis from 74 studies, encompassing 29 experimental prediction techniques, 368 guide sequences, 226164 potential guide and off-target pairs and 8840 validated off-targets. CRISPRoffT features off-target data from different CRISPR approaches (knockout, base editing and prime editing) applied under diverse experimental conditions, including 85 different Cas/guide RNA (gRNA) combinations used across 34 different human and mouse cell lines. CRISPRoffT provides results of comparative analyses for individual guide sequences, genes, cell types, techniques and Cas/gRNA combinations under different conditions. CRISPRoffT is a unique resource providing valuable insights that facilitate the safety-driven design of CRISPR-based therapeutics, inform experimental design, advance the development of computational off-target prediction algorithms and guide RNA design algorithms.

CRISPR–Cas Systems Associated with Electrolyte-Gated Graphene-Based Transistors: How They Work and How to Combine Them

In this review, recent advances in the combination of CRISPR–Cas systems with graphene-based electrolyte-gated transistors are discussed in detail. In the first part, the functioning of CRISPR–Cas systems is briefly explained, as well as the most common ways to convert their molecular activity into measurable signals. Other than optical means, conventional electrochemical transducers are also developed. However, it seems that the incorporation of CRISPR/Cas systems into transistor devices could be extremely powerful, as the former provides molecular amplification, while the latter provides electrical amplification; combined, the two could help to advance in terms of sensitivity and compete with conventional PCR assays. Today, organic transistors suffer from poor stability in biological media, whereas graphene materials perform better by being extremely sensitive to their chemical environment and being stable. The need for fast and inexpensive sensors to detect viral RNA arose on the occasion of the COVID-19 crisis, but many other RNA viruses are of interest, such as dengue, hepatitis C, hepatitis E, West Nile fever, Ebola, and polio, for which detection means are needed.

Rapid chromosome evolution and acquisition of thermosensitive stochastic sex determination in nematode androdioecious hermaphrodites

The factors contributing to evolution of androdioecy, the coexistence of hermaphrodites and males such as in Caenorhabditis elegans, remains poorly known. However, nematodes exhibit androdioecy in at last 13 genera with the predatory genus Pristionchus having seven independent transitions towards androdioecy. Nonetheless, associated genomic architecture and sex determination mechanisms are largely known from Caenorhabditis. Here, studying 47 Pristionchus species, we observed repeated chromosome evolution which abolished the ancestral XX/XO sex chromosome system. Two phylogenetically unrelated androdioecious Pristionchus species have no genomic differences between sexes and mating hermaphrodites with males resulted in hermaphroditic offspring only. We demonstrate that stochastic sex determination is influenced by temperature in P. mayeri and P. entomophagus, and CRISPR engineering indicated a conserved role of the transcription factor TRA-1 in P. mayeri. Chromosome-level genome assemblies and subsequent genomic analysis of related Pristionchus species revealed stochastic sex determination to be derived from XY sex chromosome systems through sex chromosome-autosome fusions. Thus, rapid karyotype evolution, sex chromosome evolution and evolvable sex determination mechanisms are general features of this genus, and represent a dynamic background against which androdioecy has evolved recurrently. Future studies might indicate that stochastic sex determination is more common than currently appreciated.

TtAgo-coupled-multiplex-digtal-RPA-CRISPR/Cas12a (TCMDC) for EGFR mutations detection

Epidermal Growth Factor Receptor (EGFR) is an important target for the early evaluation, treatment, and postoperative follow-up in non-small cell lung cancer (NSCLC). Current detection technologies suffer from extended detection time and high rate of false positive amplification. Therefore, the development of rapid, highly sensitive and specific detection methods is of great significance for improving the diagnosis and treatment of lung cancer. In this study, we proposed a fast and sensitive detection method termed Thermus thermophilus Argonaute (Ttago)-Coupled-Multiplex-digital-recombinase polymerase amplification (RPA)-Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (TCMDC) detection method, integrating EGFR mutation template enrichment. Based on the cleavage principle of TtAgo, the wild type (WT) template was enriched under the action of double-guide DNA. Two CRISPR RNAs, not restricted by protospacer adjacent motif (PAM) sites, were introduced to target EGFR genes. By combining RPA with CRISPR-Cas12a, we established a single-pot, ultra-sensitive (1 copy, 0.1 %), and visually detectable method for EGFR detection. We further verified the feasibility of this approach using clinical serum samples from lung cancer patients, achieving rapid (within 1 h) and visual detection of EGFR, thereby presenting a promising clinical tool for the detection of lung cancer.

Agrobacterium-Mediated Transformation and Targeted Mutagenesis Using SpCas12f in Rice.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency. Moreover, CRISPR-Cas12f can be useful for virus vector-mediated genome editing because the size of the transgene is the major restriction in the use of virus vectors. Here, we describe our protocol for targeted mutagenesis using Cas12f derived from Syntrophomonas palmitatica (SpCas12f) via Agrobacterium-mediated transformation in rice. We also summarize some approaches to improve the frequency of targeted mutagenesis using SpCas12f.

CRISPR-Cas14a and allosteric transcription factors empowered cell-free electrochemical biosensor for highly sensitive and stable detection of progesterone in multiple scenarios

In this study, a cell-free electrochemical assay based on allosteric transcription factors (aTFs) and CRISPR-Cas14a was developed for the detection of progesterone in trace samples. This electrochemical biosensor helps to overcome the drawbacks of the traditional fluorescence assay based on the CRISPR-Cas system and aTFs combined for non-nucleic acid targets that is poorly effective for the detection of colored samples. By comparing and optimizing the concentration and length of the probes in the straight chain and hairpin structure, the sensor performance was improved. In addition, different sgRNA from other studies was designed to overcome the effect of sequence folding in the space region on Cas14a activation. Based on these optimization results, we constructed an electrochemical sensor for progesterone quantification in the range of 66.7pM to 3.33 × 10−1μM. This method requires only 2 μL of sample and does not necessitate complex pretreatment steps, with detection completed within 1.5 h. The method has been successfully applied to food, environmental, and biological samples, with recovery rates between 82.65% and 109%. This suggests that CRISPR and allosteric transcription factor-powered electrochemical detection methods have significant potential for use in the field of small molecule detection under various scenarios.

CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications

Pigs have long been integral to human society for their roles in agriculture and medicine. Consequently, there is an urgent need for genetic improvement of pigs to meet human dual needs for medicine and food. In agriculture, gene editing can improve productivity traits, such as growth rate and disease resistance, which could lower farming costs and benefit consumers through enhanced meat quality. In biomedical research, gene-edited pigs offer invaluable resources as disease models and in xenotransplantation, providing organs compatible with human physiology. Currently, with CRISPR technology, especially the CRISPR/Cas9 system emerging as a transformative force in modern genetics, pigs are not only sources of sustenance but also cornerstones of biomedical innovation. This review aims to summarize the applications of CRISPR/Cas9 technology in developing pigs that serve dual roles in agriculture and biomedical applications. Compared to ZFNs and TALENs, the CRISPR/Cas9 system offers several advantages, including higher efficiency, greater specificity, ease of design and implementation, and the capability to target multiple genes simultaneously, significantly streamlining the process of genetic modifications in complex genomes. Therefore, CRISPR technology supports the enhancement of traits beneficial for agricultural productivity and facilitates applications in medicine. Furthermore, we must acknowledge the inherent deficiencies and technical challenges of the CRISPR/Cas9 technology while also anticipating emerging technologies poised to surpass CRISPR/Cas9 as the next milestones in gene editing. We hypothesize that with the continuous advancements in gene editing technologies and successful integration of traits beneficial to both agricultural productivity and medical applications, the goal of developing dual-purpose pigs for both agricultural and medical use can ultimately be achieved.

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.