Improved gene editing and fluorescent-protein tagging in using a Golden Gate-based CRISPR-Cas9 plasmid system.

CRISPR-Cas9 systems can be used for precise genome editing in filamentous fungi, including . However, current CRISPR-Cas9 systems for rely on relatively complex or multi-step cloning methods to build a plasmid expressing both Cas9 and an sgRNA targeting a genomic locus. In this study we improve on existing plasmid-based CRISPR-Cas9 systems for by creating an extremely simple-to-use CRISPR-Cas9 system for genome editing. In our system, a plasmid containing both Cas9 and an sgRNA is assembled in a one-step Golden Gate reaction. We demonstrate precise, scarless genome editing with nucleotide-level DNA substitutions, and we demonstrate markerless gene tagging by fusing fluorescent-protein coding sequences to the endogenous coding sequences of several genes. We also describe codon-adjusted versions of multiple recent-generation fluorescent proteins, which will be useful to the wider community.