CRISPR Research Articles

Structural basis of Cas3 activation in type I-C CRISPR-Cas system.

CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.

One-tube target amplification-free CRISPR-Cas13 autocatalytic system

Clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics have shown great promise for nucleic acid testing. However, CRISPR/Cas13a-based RNA sensing is hindered by the absence of accurate, fast and sensitive assays. Current Cas-based methods generally involve two separate steps: target-amplification and Cas-detection. This two-step method complicates detection, causes cross-contamination, and is difficult to deploy. Therefore, it is necessary to develop a facile one-step Cas13a-based RNA detection assay. We present a one-tube target amplification-free CRISPR-Cas13a autocatalytic circuit system that combines the Cas13a nuclease, T4 polynucleotide kinase, Bsu DNA polymerase and T7 RNA polymerase with a universally designed combinatorial DNA probe to create a one-pot assay for isothermal RNA detection. In this assay, after target RNA recognition, the cis-cleavage and trans-cleavage activities of Cas13 are activated, which leads to the initiation of a Cas13a autocatalytic reaction, resulting in an exponentially amplifying detection signal, thus enabling the detection of approximately 23.01 (95 % CI:16.95-41.23) copies/µL RNA in 45 min. A facile, easy-to-deploy, and one-tube Cas13a-based autocatalytic reaction system was constructed for rapid and highly sensitive RNA detection for the first time. This Cas13a-based autocatalytic system offers not only a versatile platform for directly detecting RNA targets but also a route toward point-of-care nucleic acid testing.

Inhibition of IRP2-dependent reprogramming of iron metabolism suppresses tumor growth in colorectal cancer

BackgroundDysregulation of iron metabolism is implicated in malignant transformation, cancer progression, and therapeutic resistance. Here, we demonstrate that iron regulatory protein 2 (IRP2) preferentially regulates iron metabolism and promotes tumor growth in colorectal cancer (CRC).MethodsIRP2 knockdown and knockout cells were generated using RNA interference and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 methodologies, respectively. Cell viability was evaluated using both CCK-8 assay and cell counting techniques. Furthermore, IRP2 inhibition was determined by surface plasmon resonance (SPR) and RNA immunoprecipitation (IP). The suppressive effects of IRP2 were also corroborated in both organoid and mouse xenograft models, providing a comprehensive validation of IRP2’s role.ResultsWe have elucidated the role of IRP2 as a preferential regulator of iron metabolism, actively promoting tumorigenesis within CRC. Elevated levels of IRP2 expression in patient samples are correlated with diminished overall survival, thereby reinforcing its potential role as a prognostic biomarker. The functional suppression of IRP2 resulted in a pronounced delay in tumor growth. Building on this proof of concept, we have developed IRP2 inhibitors that significantly reduce IRP2 expression and hinder its interaction with iron-responsive elements in key iron-regulating proteins, such as ferritin heavy chain 1 (FTH1) and transferrin receptor (TFRC), culminating in iron depletion and a marked reduction in CRC cell proliferation. Furthermore, these inhibitors are shown to activate the AMPK-ULK1-Beclin1 signaling cascade, leading to cell death in CRC models.ConclusionsCollectively, these findings highlight the therapeutic potential of targeting IRP2 to exploit the disruption of iron metabolism in CRC, presenting a strategic advancement in addressing a critical area of unmet clinical need.

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

CRISPR/dCas12a knock-down of Acidithiobacillus ferrooxidans electron transport chain bc1 complexes enables enhanced metal sulfide bioleaching

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

Integrating high mannose-binding aptamer and CRISPR/Cas12a machinery for the remote detection of cell surface high mannose

High mannose is overexpressed on the cell surface during carcinogenesis; however, there are currently no efficient techniques for its detection. Herein, we propose an assay called RADAR (remote high mannose detection on cell surface with CRISPR) for the quantification of high mannose, leveraging the integration of high mannose-binding aptamer and CRISPR technique. The high mannose-binding aptamer is hybridized with a locker, which is released upon specific binding to high mannose. The released locker then activates CRISPR/Cas12a to produce a fluorescence signal. The "remote" concept in RADAR refers to the recognition of high mannose occurring on the cell surface, while signal generation takes place in a solution separate from the target. This feature makes RADAR suitable for the detection of suspended bio-samples without the need for sophisticated imaging. RADAR can detect high mannose Man9 with a limit of detection of 2.5 nM, distinguish cancer cells from non-cancerous cells, and profile high mannose level in human serum for potential non-small cell lung cancer (NSCLC) diagnosis. This work represents the first attempt to integrate CRISPR technology into the aptasensing of high mannose, which may serve as a synthetic biology-enabled tool in glycochemistry and glycobiology.

Development of an inducer-free, virulence gene promoter-controlled, and fluorescent reporter-labeled CRISPR interference system in Staphylococcus aureus.

The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus.IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.

CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.

Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

Establishing CRISPR-Cas9 in the sexually dimorphic moss, Ceratodon purpureus.

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.

The genetic landscape of antibiotic sensitivity in Staphylococcus aureus.

Despite the critical importance of essential genes, systems-level investigations of their contribution to antibiotic sensitivity have been limited. Using CRISPR Adaptation-mediated Library Manufacturing (CALM), we generated ultra-dense CRISPR interference (CRISPRi) libraries in methicillin-sensitive and -resistant strains of Staphylococcus aureus, which allowed us to quantify gene fitness on a global scale across ten clinically relevant antibiotics. This led to the identification of a comprehensive set of known and novel biological processes modulating bacterial fitness in the antibiotics. Notably, we found that essential genes from diverse processes dominated antibiotic-gene interactions, including a large number of synergistic interactions between bactericidal antibiotics and processes such as cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), protein export, and coenzyme A biosynthesis. Simultaneous genetic perturbations of diverse CC and DD processes aggravated bacterial fitness, revealing a widespread synergy between the two highly coordinated processes. In contrast, perturbation of transcriptional, translational, and select energy processes antagonized the effects of bactericidal antibiotics. Finally, we show that small molecule inhibitors recapitulated synergistic antibiotic-gene interactions, providing a rational foundation for developing novel combinatorial antimicrobial therapies.

Scanning amplicons with CRISPR-Dx detects endangered amphibians in environmental DNA.

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large-scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species-specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species-specific CRISPR-Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term 'ampliscanning', for 18 of the 22 amphibian species in Switzerland. We sub-selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR-Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR-Dx combined with universal primers for large-scale monitoring of multiple endangered species across landscapes to inform conservation measures.

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Current developments of gene therapy in human diseases.

Gene therapy has witnessed substantial advancements in recent years, becoming a constructive tactic for treating various human diseases. This review presents a comprehensive overview of these developments, with a focus on their diverse applications in different disease contexts. It explores the evolution of gene delivery systems, encompassing viral (like adeno-associated virus; AAV) and nonviral approaches, and evaluates their inherent strengths and limitations. Moreover, the review delves into the progress made in targeting specific tissues and cell types, spanning the eye, liver, muscles, and central nervous system, among others, using these gene technologies. This targeted approach is crucial in addressing a broad spectrum of genetic disorders, such as inherited lysosomal storage diseases, neurodegenerative disorders, and cardiovascular diseases. Recent clinical trials and successful outcomes in gene therapy, particularly those involving AAV and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins, are highlighted, illuminating the transformative potentials of this approach in disease treatment. The review summarizes the current status of gene therapy, its prospects, and its capacity to significantly ameliorate patient outcomes and quality of life. By offering comprehensive analysis, this review provides invaluable insights for researchers, clinicians, and stakeholders, enriching the ongoing discourse on the trajectory of disease treatment.

An Enthusiastic but Uncertain Welcome: Coverage of Risks, Benefits, and Social Contexts of CRISPR Technology in U.S. Agricultural News 2012-2022

An Enthusiastic but Uncertain Welcome: Coverage of Risks, Benefits, and Social Contexts of CRISPR Technology in U.S. Agricultural News 2012-2022

An enterococcal phage protein inhibits type IV restriction enzymes involved in antiphage defense

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

Uncovering the genomic basis of symbiotic interactions and niche adaptations in freshwater picocyanobacteria

BackgroundPicocyanobacteria 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.ResultsIn 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.ConclusionsOur findings provide novel insights into the population diversity, ecology, and evolutionary strategies of the most widespread photoautotrophs within freshwater ecosystems.62j11owDS1FAqTqkQkf7rCVideo

Less is more: CRISPR/Cas9-based mutations in DND1 gene enhance tomato resistance to powdery mildew with low fitness costs

Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.