Establishment of an Efficient CRISPR-Cas9-Mediated Gene Disruption System in the Lichen-Forming Fungus Umbilicaria muhlenbergii

Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint

A substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control is revealed, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.

A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR/Cas9-Based Editing System in the Filamentous Fungus Fusarium oxysporum

Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA−sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR/Cas9 system exhibited a high efficiency of 93.75% ± 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (∼1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (∼19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, ∼1.5 kb) and pacC (pH-responsive transcription factor, ∼2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.

Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola ‘Sunset Velvet’

Homologous knock-in processes facilitated by CRISPR-Cas9 in Aurantiochytrium and disrupting the gene for DNA ligase IV prevents transformation events

The potential of these CRISPR-Cas9 systems to serve as a robust foundation for the functional genomics and metabolic engineering of A. limacinum is demonstrated.

CRISPR/Cas9-mediated transformation enables functional characterization of the effector Avr4 in the banana pathogen Pseudocercospora fijiensis

It is demonstrated that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors.

Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.