Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality

Abstract

Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.

Synthetic biology for Monascus: From strain breeding to industrial production

“…[75] At the same time, filamentous fungi lack certain promoters necessary for expressing sgRNA, making it difficult for the CRISPR-Cas9 system to proceed smoothly in filamentous fungi. [29] Consequently, researchers have reasonably adjusted the CRISPR-Cas system, forming a stan-dardized set of CRISPR editing processes for filamentous fungi. This adaptation has sparked significant interest in applying this gene editing system in filamentous fungi (Figure 3).…”

“…[26,27] Compared to ZFN and TALEN technologies, gene editing technology using CRISPR/Cas9 shows the advantages of simple operation, good targeting, and high safety, which helps it become the preferred technology for gene manipulations of filamentous fungi. [28,29] This article summarizes the most commonly used techniques in the metabolic engineering of filamentous fungi, followed by an overview of the current industrial production strains of MP and lovastatin and the elimination of citrinin (CIT). We also discuss the key issues that occurred in the metabolic engineering of Monascus to provide a theoretical basis for applying synthetic biotechnology in the metabolism of filamentous fungi.…”

Synthetic biology tools for production of insect pheromones in plants and filamentous fungi

“…Originally discovered as part of the adaptive immune system in bacteria, CRISPR/Cas ribonucleoproteins have been repurposed as genome editing tools by harnessing their remarkably accurate ability to target specific DNA sequences. The fast development of CRISPR-based technologies has provided powerful and versatile tools for efficient genome editing in many organisms, including plants and filamentous fungi (Liu et al, 2022;Shen et al, 2023;Zhang et al, 2019). Contrary to other genome-editing nucleases commonly applied like Transcription activator-like effectors (TALEs) or Zinc Fingers that require the re-design of their coding sequence to target different DNA sequences (Morbitzer et al, 2010;Urnov et al, 2010), the activity of Cas nucleases is directed by means of the guide RNA (gRNA) employed, which enhances their customizability and modularity.…”