Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression

Abstract

Background: Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.

Results: The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.

Conclusion: The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Keywords: Aureobasidium pullulans; AMA1 plasmid; CRISPR/Cas9; Fungal transformation; Golden Gate Assembly; Protoplasts.

Fig. 1

Overview on the CRISPR/Cas9 plasmid assembly. A. Scheme of the multiple overlap PCR to generate the 248 bp fragment. This fragment was assembled by PCR using two primers containing the sequence specific to the gRNA target and four generic primers. The obtained product includes a hammerhead ribozyme, the complementary sequence to the target DNA, the sequence for expression of the gRNA, a hepatitis delta virus (HDV) ribozyme for cutting at the 5’ and 3’ ends of the gRNA and BsaI restriction sites on the 3’ and 5’ ends. B. Overview on the two GGAs to generate the final CRISPR/Cas9 plasmid. The initial GGA using BsaI, leads to the ligation of the 248 bp-fragment and the BB1_L_23_syn_BsaI plasmid. The product of this assembly is then assembled with the plasmid bearing the Cas9 expression cassette via BsbI

Fig. 2

Sequencing results of obtained transformants. A schematic drawing of the URA3 locus is given with the target of the CRISPR/Cas9 system. Red line and letters, PAM sequence; green line and letters, sequence complementary to the gRNA; purple letters, nucleotides being part of the URA3 locus; blue letters, nucleotides upstream and downstream of the URA3 locus; pink, italic letters, inserted nucleotides; Strain, strains analyzed; WT, wild-type; strain numbers starting with 1, transformants obtained from the first transformation; strain numbers starting with 2, transformants obtained from the second transformation; Start, genomic position in the WT. Sequence, obtained sequencing result; End, genomic position in the WT; Del, number of nucleotides deleted

Fig. 3

Phenotype of selected transformants. Growth of the wild-type and obtained transformants was tested on plates with the indicated medium. From the first transformation assay (row 1) the transformant 1.2 (∆URA) and the WT are displayed. From the second transformation (row 2) the 33 selected transformants were tested and are displayed together with the wild-type (WT), the URA3 mutant strain #6 from [19] (#6), and an A. pullulans EXF-150 double deletion mutant with a hygromycin B genomic resistance cassette (∆∆). Tran number of transformations, MEX malt extract, uri uridine, hyg hygromycin B, 5-FOA 5-fluoroorotic acid