Research progress of CRISPR/Cas9 system in genome targeted editing of filamentous fungi

Abstract:

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology, established in 2013, guided by special RNA and initiated by endonuclease, has been developed into the third generation of genome editing technology, which is widely used in genome editing among varieties of species including animals, plants, bacteria and fungi with high efficiency and specificity. This review briefly introduces the characteristics and the application of three genome editing technologies based on endonucleases, describes the composes and mechanism of CRISPR/Cas9 mediated genome editing, summarizes the application of this tool in Saccharomyces cerevisiae and filamentous fungi in genome editing. In the other hand, we put forward the protocol about using CRISPR/Cas9 technology in filamentous fungi, such as the design of sgRNA cassette, the optimization of Cas9 cassette, the screening of resistance marker, the selection of receptor, and so on. Furthermore, some suggestions were given about the problems which often encounter in practical application, such as off-target effects, the addition of Cas9 nuclear localization signal, the selection of promoter, and multi-gene editing, to provide a theoretical and technological references for the beginners in filamentous fungus gene editing field.