Progress and Challenges: Development and Implementation of CRISPR/Cas9 Technology in Filamentous Fungi.

Widely distributed in various environmental niches, filamentous fungi play an important role in industry, drug development, and plant/animal health. Manipulation of the genome and the coding sequences are essential for a better understanding of the function of genes and their regulation, but traditional genetic approaches in some filamentous fungi are either inefficient or nonfunctional. The rapid development and wide implementation of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats /(CRISPR)-associated protein-9 nuclease) technology for various model and non-model organisms has provided the initial framework to adapt this gene editing technology for filamentous fungi. In this review, an overview of the CRISPR/Cas9 tools and strategies that have been developed for different filamentous fungi is presented, including integration of the gene into the genome, transient expression of Cas9/sgRNA, the AMA1-based plasmid approach, and the Cas9 RNP method. The various applications of CRISPR/Cas9 technology in filamentous fungi that have been implemented are explored, with particular emphasis on gene disruption/deletion and precise genome modification through gene tagging and alteration in gene regulation. Potential challenges that are confronted when developing a CRISPR/Cas9 system for filamentous fungi are also discussed such as the nuclear localization sequence for the gene, potential off-target effects, and highly efficient transformation methods. Overcoming these obstacles may further facilitate wide application of this technology. As a simple, economical, and powerful tool, CRISPR/Cas9 systems have the potential for future implementation into many molecular aspects of filamentous fungi.