Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

Theranostics, Год журнала: 2020, Номер 11(2), С. 614 - 648

Опубликована: Окт. 28, 2020

CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy been hindered by efficient delivery materials target cells. Direct system as a ribonucleoprotein (RNP) complex consisting Cas9 protein and single guide RNA (sgRNA) emerged powerful widespread method for due to its advantages transient reduced off-target effects. In review, we summarized current RNP systems including physical approaches synthetic carriers. The mechanisms beneficial roles these strategies intracellular were reviewed. Examples development stimuli-responsive targeted carriers are highlighted. Finally, challenges perspectives rational design next generation promising field will be discussed.

Язык: Английский

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Killing softly: a roadmap of Botrytis cinerea pathogenicity

Trends in Plant Science, Год журнала: 2022, Номер 28(2), С. 211 - 222

Опубликована: Сен. 30, 2022

Botrytis cinerea, a widespread plant pathogen with necrotrophic lifestyle, causes gray mold disease in many crops. Massive secretion of enzymes and toxins was long considered to be the main driver infection, but recent studies have uncovered rich toolbox for B. cinerea pathogenicity. The emerging picture is multilayered infection process governed by exchange factors that collectively contribute development. No shows complete resistance against pattern-triggered immune responses potential significantly reduce progression, opening new possibilities producing cinerea-tolerant plants. We examine current models, highlight knowledge gaps, suggest directions future studies.

Язык: Английский

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Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea

PLoS Pathogens, Год журнала: 2022, Номер 18(3), С. e1010367 - e1010367

Опубликована: Март 3, 2022

Botrytis cinerea is a major plant pathogen infecting more than 1400 species. During invasion, the fungus rapidly kills host cells, which believed to be supported by induction of programmed cell death. To comprehensively evaluate contributions most currently known death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established allowed perform serial marker-free mutagenesis generate multiple deletion mutants lacking up 12 CDIPs. Whole genome sequencing 6x 12x mutant revealed low number off-target mutations were unrelated Cas9-mediated cleavage. Secretome analyses confirmed loss secreted encoded deleted genes. Infection tests with successive decrease in virulence increasing numbers mutated genes, varying effects knockouts on different plants. Comparative analysis significant roles two polygalacturonases (PG1, PG2) phytotoxic botrydial botcinins but no or only weak other Nicotiana benthamiana plants silenced coreceptors pattern recognition receptors, SOBIR1 BAK1, showed similar susceptibility as control infection B. wild type mutant. These results raise doubts about role manipulation these defence regulators infection. Despite compounds, planta secretomes retained substantial activity, proving that further, yet unknown CDIPs contribute necrosis virulence. Our study has addressed first time systematically functional redundancy fungal factors, demonstrates releases highly redundant cocktail achieve wide variety

Язык: Английский

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Plant pathogenic fungi hijack phosphate signaling with conserved enzymatic effectors

Science, Год журнала: 2025, Номер 387(6737), С. 955 - 962

Опубликована: Фев. 27, 2025

Inorganic phosphate (Pi) is essential for life, and plant cells monitor Pi availability by sensing inositol pyrophosphate (PP-InsP) levels. In this work, we describe the hijacking of a conserved family Nudix hydrolase effectors from pathogenic Magnaporthe Colletotrichum fungi. Structural enzymatic analyses effector demonstrate that they selectively hydrolyze PP-InsP. Gene deletion experiments in oryzae, higginsianum, graminicola indicate PP-InsP hydrolysis substantially enhances disease symptoms diverse pathosystems. Further, show induces starvation signaling plants. Our study elucidates molecular mechanism, used multiple phytopathogenic fungi, manipulates highly pathway to exacerbate disease.

Язык: Английский

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The Destructive Fungal Pathogen Botrytis cinerea—Insights from Genes Studied with Mutant Analysis

Pathogens, Год журнала: 2020, Номер 9(11), С. 923 - 923

Опубликована: Ноя. 7, 2020

Botrytis cinerea is one of the most destructive fungal pathogens affecting numerous plant hosts, including many important crop species. As a molecularly under-studied organism, its genome was only sequenced at beginning this century and it recently updated with improved gene annotation completeness. In review, we summarize key molecular studies on B. developmental pathogenesis processes, specifically genes studied comprehensively mutant analysis. Analyses these have unveiled in biological processes pathogen, hyphal growth, sclerotial formation, conidiation, pathogenicity melanization. addition, our synthesis has uncovered gaps present knowledge regarding development virulence mechanisms. We hope review will serve to enhance mechanisms behind notorious pathogen.

Язык: Английский

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Fungal biotechnology: From yesterday to tomorrow

Frontiers in Fungal Biology, Год журнала: 2023, Номер 4

Опубликована: Март 27, 2023

Fungi have been used to better the lives of everyday people and unravel mysteries higher eukaryotic organisms for decades. However, comparing progress development stemming from fungal research that human, plant, bacterial research, fungi remain largely understudied underutilized. Recent commercial ventures begun gain popularity in society, providing a new surge interest fungi, mycelia, potential applications these various aspects research. Biotechnological advancements cannot occur without intensive amounts time, investments, tool development. In this review, we highlight past breakthroughs biotechnology, discuss requirements advance biotechnology even further, touch on horizon with highest positively impact both society.

Язык: Английский

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Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

Journal of Fungi, Год журнала: 2023, Номер 9(3), С. 362 - 362

Опубликована: Март 15, 2023

Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and other industries. The genes involved SM biosynthesis are usually clustered adjacent to each into a region known as biosynthetic gene cluster (BGC). recent advent diversity genetic genomic technologies has facilitated the identification cryptic or uncharacterized BGCs their associated SMs. However, there still challenges that hamper broader exploration industrially metabolites. advanced CRISPR/Cas system revolutionized fungal engineering enabled discovery novel compounds. In this review, we firstly introduce relationships with SMs, followed by brief summary conventional strategies for engineering. Next, range state-of-the-art CRISPR/Cas-based tools been developed review these methods fungi research on Finally, limitations systems discussed directions future proposed order expand improve efficiency

Язык: Английский

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Botrytis cinerea combines four molecular strategies to tolerate membrane-permeating plant compounds and to increase virulence

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Июль 31, 2024

Saponins are plant secondary metabolites comprising glycosylated triterpenoids, steroids or steroidal alkaloids with a broad spectrum of toxicity to microbial pathogens and pest organisms that contribute basal defense biotic attack. Secretion glycosyl hydrolases enzymatically convert saponins into less toxic products was thus far the only mechanism reported enable fungal colonize their saponin-containing host plant(s). We studied mechanisms fungus Botrytis cinerea utilizes be tolerant well-characterized, structurally related from tomato Digitalis purpurea. By gene expression studies, comparative genomics, enzyme assays testing large panel (knockout complemented) mutants, we unraveled four distinct cellular participate in mitigation activity these virulence on saponin-producing plants. The enzymatic deglycosylation identified is novel unique this fungus-saponin combination. other three tolerance operate membrane mediated by protein families widely distributed kingdom. present spatial temporal model how jointly confer discuss repercussions findings for pathogenic fungi, as well human pathogens. Plant antimicrobial toxicity. Here, You et al. characterize which establishes saponins.

Язык: Английский

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Recent advances in the application of CRISPR/Cas-based gene editing technology in Filamentous Fungi

Biotechnology Advances, Год журнала: 2025, Номер 81, С. 108561 - 108561

Опубликована: Март 12, 2025

Язык: Английский

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The contribution of DNA repair pathways to genome editing and evolution in filamentous pathogens

FEMS Microbiology Reviews, Год журнала: 2022, Номер 46(6)

Опубликована: Июль 9, 2022

DNA double-strand breaks require repair or risk corrupting the language of life. To ensure genome integrity and viability, multiple break pathways function in eukaryotes. Two such pathways, canonical non-homologous end joining homologous recombination, have been extensively studied, while other as microhomology-mediated joint single-strand annealing, once thought to serve back-ups, now appear play a fundamental role repair. Here, we review molecular details hierarchy these four where possible, comparison for what is known between animal fungal models. We address factors contributing pathway choice, aim explore our understanding knowledge gaps regarding mechanisms regulation filamentous pathogens. additionally discuss how influence engineering results, including unexpected mutation outcomes. Finally, concept biased evolution pathogens, provide model, termed Biased Variation, that links with properties evolution. Despite extensive this universal process, there remain many unanswered questions, which answers may improve

Язык: Английский

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