System overview of genomic edition in N. crassa using CRISPR/Cas9. a The system consists of two components, a Cas9 protein and a single crRNA:tracrRNA chimeric guide RNA (gRNA), comprising a 20-bp target sequence (red) complementary to the genomic target adjacent to a PAM site of NGG (blue). b Design of the Cas9 and gRNA constructs. The Cas9 protein contained a SV40 nuclear localization signal, and the expression was under the control of the trpC promoter and terminator. The gRNA was expressed under the snoRNA SNR52 promoter and contained a terminator from the 30 region of the yeast SUP4 gene. c Design of gRNA targeted to clr-2 and csr-1 loci.