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.

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

“…The Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system has brought a remarkable development in genome engineering efficiency in different organisms during the past few years (Doudna and Charpentier, 2014;Albadri et al, 2017;Ermert et al, 2019;Pu et al, 2019;Song et al, 2019). Briefly, the CRISPR-cas system employs a guide RNA (gRNA) and an endonuclease, mostly a single nuclease Cas9 (Makarova et al, 2011;Chylinski et al, 2014), as the two working elements for a site directed DNA cutting.…”

Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

“…Multiplex gene modification methods, as demonstrated in this work, enables the modification of several genes simultaneously and permits a rapid generation of engineered strains. CRISPR/Cas9 technologies has been described for genome editing of many filamentous fungi using different strategies for vector construction and transformation methods [11]. In contrast, Cpf1-based systems have been only described for two Aspergilli species among filamentous fungi [21].…”

“…The CRISPR/Cas9 systems have emerged as the foremost technique for genome engineering of many organisms, including yeasts and fungi, with applications that go further beyond the single gene modification (deletions and nucleotide substitutions) [10]. Thus, gene regulation and systems metabolic engineering approaches have been described using CRISPR/Cas9 systems in different yeasts and fungi [10,11].…”

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

“…We centred our approach on a dCas9-based gene activation system because with this, the activator can be targeted at one or more genomic locations easily and simultaneously simply by expressing the right combination of guide RNAs. Moreover, the functionality of the conventional CRISPR/Cas9 system for genetic manipulation of a single site or multiple loci (multiplex sgRNAs) is firmly established in A. nidulans and other filamentous fungi as reviewed by Song et al (Nodvig et al, 2018;Song et al, 2019). We reasoned that VPR-dCas9 can be expressed in the host and guided by co-expressed sgRNAs to any accessible locus in the genome.…”