1. Editorial : Mechanisms of plant host resistance against virusesRégis L. Corrêa, Marko Petek, Maite F. S. Vaslin, 2024, drugi znanstveni članki Ključne besede: plants, pathogens, plant viruses, resistance, biotechnological tools, CRISPR/Cas9, agriculture, biotechnology
Objavljeno v DiRROS: 05.11.2024; Ogledov: 185; Prenosov: 52
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2. CRISPR/Cas-mediated plant genome editing : outstanding challenges a decade after implementationTeodoro Cardi, Jana Murovec, Allah Bakhsh, Vladislava Galović, Tjaša Lukan, Kubilay Yıldırım, Milica Zlatković, Katrijn Van Laere, 2023, pregledni znanstveni članek Povzetek: The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
Ključne besede: CRISPR applications, CRISPR platforms, gene regulations, mutant detection, plant regeneration
Objavljeno v DiRROS: 05.08.2024; Ogledov: 254; Prenosov: 252
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3. Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces speciesAlen Pšeničnik, Roman Reberšek, Lucija Slemc, Tim Godec, Luka Kranjc, Hrvoje Petković, 2022, izvirni znanstveni članek Povzetek: CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
Ključne besede: CRISPR-Cas9, gusA visual screening, "CRISPR escaper colonies", Streptomyces
Objavljeno v DiRROS: 17.07.2024; Ogledov: 273; Prenosov: 209
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4. CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potatoTjaša Lukan, Florian Veillet, Maja Križnik, Anna Coll Rius, Tjaša Mahkovec Povalej, Karmen Pogačar, Katja Stare, Laura Chauvin, Jean-Eric Chauvin, Kristina Gruden, 2022, izvirni znanstveni članek Povzetek: MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.
Ključne besede: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Objavljeno v DiRROS: 17.07.2024; Ogledov: 337; Prenosov: 227
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