CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective

Abstract

Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.

Keywords: CRISPR/Cas9; Filamentous fungi; Genome editing; Off-target.

Fig. 1

Schematic illustration of Cas9/gRNA genome editing. a sgRNA-mediated Cas9 protein can bind to target sequences of site and cut DNA double strands. b When the DNA double-strand breaks occur, the cell initiates the self-repair mechanism. The NHEJ-dominated repair pathway will cause the random loss, insertion, and replacement of bases at the breakage point, resulting in gene mutation. The HR pathway will accurately edit the target gene guided by the donor DNA fragment. c Single-promoter-driven gRNA expression cassette. d multiple gRNA expression cassettes can be constructed by concatenating 2 or more of gRNA linked together by linkers, which can then be enzymatically processed into multiple single gRNAs thus targeting multiple sites

Fig. 2

The history of the development and application of CRISPR/Cas9 technology in filamentous fungi; different colors represent different promoter-driven gRNA expression cassettes