Potassium (K+) is an essential macronutrient which contributes to osmotic- and turgor-related processes in plants. Calcineurin-B like Interacting Protein Kinases (CIPKs) play crucial roles in plants under low-K+ supply since they activate root K+ uptake transport systems such as AKT1 and AtHAK5. In Arabidopsis, AtCIPK9 is important for low-K+ tolerance since atcipk9 plants exhibited poor growth and leaf chlorosis when K+ was scarce. Part of these phenotypes could be ascribed to the activation of AtHAK5 by AtCIPK9. It has been reported that important differences exist between Arabidopsis and other plant species such as tomato with respect to the regulation of K+ uptake systems. Thus, our aim was to evaluate the contribution of SlCIPK9, the homologous protein of AtCIPK9 in tomato, to K+ nutrition. Unexpectedly, phenotyping experiments carried out with slcipk9 loss-of-function mutants revealed that SlCIPK9 did not play a clear role in tomato K+ homeostasis. By contrast, it was found that SlCIPK9 contributed to pollen tube elongation, but not to pollen germination, via a K+-independent mechanism. Therefore, our results highlight the remarkable differences that exist in Ca2+ signaling pathways between plant species and encourage the realization of more comparative studies as the one presented here.

The global burden of cancer and the limitations of conventional therapies highlight the potential of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) in reshaping cancer treatment paradigms. In this review, we have investigated the mechanism of CRISPR, an adaptive immune system in bacteria that enables highly precise gene editing at the molecular level. This versatile tool demonstrates its efficacy in human cancer therapy through gene knockout, metabolic disruption, base editing, screening, and immunotherapy enhancement without affecting normal bodily domains. Despite its superiority over other nucleases like zinc-finger nucleases and transcription activator-like effector nucleases, hurdles such as off-target effects, inefficient delivery of the system to target cells, the emergence of escapers, and the ethical debate surrounding genome editing are discussed. In this article, we have reviewed the promising approaches of CRISPR-Cas9 in cancer treatment while exploring the underlying mechanism, advantages, and associated challenges.

BACKGROUND: Picocyanobacteria from the genera Prochlorococcus, Synechococcus, and Cyanobium are the most widespread photosynthetic organisms in aquatic ecosystems. However, their freshwater populations remain poorly explored, due to uneven and insufficient sampling across diverse inland waterbodies.
RESULTS: In this study, we present 170 high-quality genomes of freshwater picocyanobacteria from non-axenic cultures collected across Central Europe. In addition, we recovered 33 genomes of their potential symbiotic partners affiliated with four genera, Pseudomonas, Mesorhizobium, Acidovorax, and Hydrogenophaga. The genomic basis of symbiotic interactions involved heterotrophs benefiting from picocyanobacteria-derived nutrients while providing detoxification of ROS. The global abundance patterns of picocyanobacteria revealed ecologically significant ecotypes, associated with trophic status, temperature, and pH as key environmental factors. The adaptation of picocyanobacteria in (hyper-)eutrophic waterbodies could be attributed to their colonial lifestyles and CRISPR-Cas systems. The prevailing CRISPR-Cas subtypes in picocyanobacteria were I-G and I-E, which appear to have been acquired through horizontal gene transfer from other bacterial phyla.
CONCLUSIONS: Our findings provide novel insights into the population diversity, ecology, and evolutionary strategies of the most widespread photoautotrophs within freshwater ecosystems. Video Abstract.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated nuclease (Cas) system has been proven to play an irreplaceable role in bacteria immunity activity against exogenous genetic elements. In recent years, this system has emerged as a valid gene engineering method and could be used to detect and treat various microorganisms such as bacteria and viruses, etc. Staphylococcus aureus, as a Gram-positive, opportunistic human and animal pathogen, can cause a variety of diseases greatly threatening human health. Here, we mainly reviewed the applications of the CRISPR-Cas system in Staphylococcus aureus infections in detail. Furthermore, the prospects and drawbacks of the CRISPR-Cas system were also discussed.

CRISPR-associated proteins and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) technology has emerged as a groundbreaking advancement in animal and poultry nutrition to improve feed conversion efficiency, enhance disease resistance, and improve the nutritional quality of animal products. Despite significant advancements, there is a research gap in the systematic understanding and comprehensive use of the CRISPR-Cas method in animal and poultry nutrition. The purpose of this study is to elucidate the latest advancements in animal and poultry nutrition through CRISPR-Cas genome editing technology, focusing on gene manipulation in metabolism, immunity, and growth. Following preferred reporting items in meta-analysis and systematic reviews guidelines, we conducted a systematic search using several databases, including Scopus, PubMed, and Web of Science, until May 2024, and finally, we included a total of 108 articles in this study. This article explores the use of the CRISPR-Cas system in the advancement of feed additives like probiotics and enzymes, which could reduce the use of antibiotics in animal production. Furthermore, the article discusses ethical and regulatory issues related to gene editing in animal and poultry nutrition, including concerns about animal welfare, food safety, and environmental impacts. Overall, the CRISPR-Cas system holds substantial promise to overcome the challenges in modern animal agriculture. By enriching the nutritional quality of animal products, increasing disease resistance, and improving feed efficiency, it offers sustainable and cost-effective solutions that can revolutionize animal and poultry nutrition.

The constant arms race between bacteria and their parasites has resulted in a large diversity of bacterial defenses, with many bacteria carrying multiple systems. Here, we report the discovery of a phylogenetically widespread defense system, coined methylation-associated defense system (MADS), which is distributed across gram-positive and gram-negative bacteria. MADS interacts with a CRISPR-Cas system in its native host to provide robust and durable resistance against phages. While phages can acquire epigenetic-mediated resistance against MADS, co-existence of MADS and a CRISPR-Cas system limits escape emergence. MADS comprises eight genes with predicted nuclease, ATPase, kinase, and methyltransferase domains, most of which are essential for either self/non-self discrimination, DNA restriction, or both. The complex genetic architecture of MADS and MADS-like systems, relative to other prokaryotic defenses, points toward highly elaborate mechanisms of sensing infections, defense activation, and/or interference.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 μL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

Advancements in the CRISPR technology, a game-changer in experimental research, have revolutionized various fields of life sciences and more profoundly, cancer research. Cell death pathways are among the most deregulated in cancer cells and are considered as critical aspects in cancer development. Through decades, our knowledge of the mechanisms orchestrating programmed cellular death has increased substantially, attributed to the revolution of cutting-edge technologies. The heroic appearance of CRISPR systems have expanded the available screening platform and genome engineering toolbox to detect mutations and create precise genome edits. In that context, the precise ability of this system for identification and targeting of mutations in cell death signaling pathways that result in cancer development and therapy resistance is an auspicious choice to transform and accelerate the individualized cancer therapy. The concept of personalized cancer therapy stands on the identification of molecular characterization of the individual tumor and its microenvironment in order to provide a precise treatment with the highest possible outcome and minimum toxicity. This study explored the potential of CRISPR technology in precision cancer treatment by identifying and targeting specific cell death pathways. It showed the promise of CRISPR in finding key components and mutations involved in programmed cell death, making it a potential tool for targeted cancer therapy. However, this study also highlighted the challenges and limitations that need to be addressed in future research to fully realize the potential of CRISPR in cancer treatment.

Klebsiella variicola is a Gram-negative bacterium that is frequently isolated from a wide variety of natural niches. It is a ubiquitous opportunistic pathogen that can cause diverse infections in plants, animals, and humans. It also has significant biotechnological potential. However, due to the lack of efficient genetic tools, the molecular basis contributing to the pathogenesis and beneficial activities of K. variicola remains poorly understood. In this study, we found and characterized a native type I-E CRISPR-Cas system in a recently isolated K. variicola strain KV-1. The system cannot cleave target DNA sequences due to the inactivation of the Cas3 nuclease by a transposable element but retains the activity of the crRNA-guided Cascade binding to the target DNA sequence. A targeting plasmid carrying a mini-CRISPR to encode a crRNA was designed and introduced into the KV-1 strain, which successfully repurposed the native type I-E CRISPR-Cas system to inhibit the expression of the target gene efficiently and specifically. Moreover, by creating a mini-CRISPR to encode multiple crRNAs, multiplex gene repression was achieved by providing a single targeting plasmid. This work provides the first native CRISPR-Cas-based tool for programmable multiplex gene repression in K. variicola, which will facilitate studying the pathogenic mechanism of K. variicola and enable metabolic engineering to produce valuable bioproducts.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single-guide RNA (sgRNA)-based gene editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, highly instructive, and inspiring for future advances in medicine. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent β-thalassemia has renewed hope for treating other hematologic diseases, including patients with a germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-inspired gene therapies. The purpose of this paper is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy, preceded by a brief description of the current tools being used in clinical genome editing; and third, a presentation of the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with a germline predisposition to hematologic malignancies.