Fig. 2

Different RNA polymerase III-based promoters for CRISPR/Cas9 systems mediated albA gene disruption in A. niger. a Sequence alignment of the promoter sequences of Homo sapiens RNU6-1, yeast RNU6, and A. niger RNU6. + 1 represents the transcription start; the TATA-like box and proximal and distal sequence elements are represented by a red box. b Schematic diagram of albA disruption mediated by NHEJ using the CRISPR/Cas9 system based on the U6 promoter. hU6 promoter represents the promoter of the human RNU6-1 gene (NR_004394); the yU6 promoter represents the promoter of the yeast RNU6 gene (X12565.1); the anU6 promoter represents the 412-bp upstream of A. niger RNU6 gene (AY136823.1). T₆ represents a string of six thymines serving as an RNA polymerase III terminator. Linear sgRNA constructs and Cas9 expression plasmid pCas9 were co-transformed into the protoplast. Without the donor DNA, the DSBs induced by Cas9 were repaired by the error-prone NHEJ system, which resulted in albA disruption. c Transformants growing on the primary transformation plates after 5 days incubation after being co-transformed with pCas9 and sgRNA expression cassettes. If albA was disrupted, the conidia of transformants turned pigmentless, forming albino colonies, as the red arrows indicate. The histogram shows the albA gene disruption efficiency of the transformants with sgRNA constructs under the control of different U6 promoters. Bars represent the percentages of albino colonies that showed the albA disruption phenotype on the primary transformation plates (mean ± SD; n = 3)