Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides

Abstract

Mucor circinelloides and other members of Mucorales are filamentous fungi, widely used as model organisms in basic and applied studies. Although genetic manipulation methods have been described for some Mucoral fungi, construction of stable integrative transformants by homologous recombination has remained a great challenge in these organisms. In the present study, a plasmid free CRISPR-Cas9 system was firstly developed for the genetic modification of a Mucoral fungus. The described method offers a rapid but robust tool to obtain mitotically stable mutants of M. circinelloides via targeted integration of the desired DNA. It does not require plasmid construction and its expression in the recipient organism. Instead, it involves the direct introduction of the guide RNA and the Cas9 enzyme and, in case of homology directed repair (HDR), the template DNA into the recipient strain. Efficiency of the method for non-homologous end joining (NHEJ) and HDR was tested by disrupting two different genes, i.e. carB encoding phytoene dehydrogenase and hmgR2 encoding 3-hydroxy-3-methylglutaryl-CoA reductase, of M. circinelloides. Both NHEJ and HDR resulted in stable gene disruption mutants. While NHEJ caused extensive deletions upstream from the protospacer adjacent motif, HDR assured the integration of the deletion cassette at the targeted site.

Figure 1

Colonies of Mucor circinelloides carB disruption mutants obtained by the CRISPR-Cas9 method. Panel A, selection of the transformed colonies obtained from the wild-type strain, CBS227.49 using NHEJ mutagenesis based on their white colony color. Panel B, colony color of the strain CBS227.49 and its carB′ disruption mutants (CBS227.49-carB′/1 and CBS227.49-carB′/2) created by NHEJ mutagenesis. Panel C, colony color of the strain MS12 and its carB disruption mutants created by NHEJ (MS12-carB′/1 and MS12-carB′/2) and HDR (MS12-carB + pyrG/1 and MS12-carB + pyrG/2) approaches.

Figure 2

Genome editing strategy designed to disrupt the carB gene of Mucor circinelloides by the CRISPR-Cas9 method and positions of the primers used in the study (A) and PCR analysis of the transformants using the MccarB1 and MccarB2 primers (Table 2) (B). TGG indicates the PAM sequence; arrows show the orientations of the primers. Panel B: M, GeneRuler 1 kb DNA ruler (Thermo Scientific); 1, MS12 strain; 2, MS12-carB + pyrG/1 strain; 3, MS12- carB + pyrG/2 strain.

Figure 3

Genome editing strategy designed to disrupt the hmgR2 gene of Mucor circinelloides by the CRISPR-Cas9 method and positions of the primers used in the study (A) and PCR analysis of the transformants using the H2cDNS1 and H2cDNS8 primers (Table 2) (B). TGG indicates the PAM sequence; arrows show the orientations of the primers. Panel B: M, GeneRuler 1 kb DNA ruler (Thermo Scientific); 1, MS12 strain; 2, MS12-hmgR2 + pyrG strain.

Figure 4

Growth ability of the mutants obtained from the Mucor circinelloides MS12 strain at 35 °C. Strains were cultivated on YNB medium for four days under continuous light. Values followed by * and ** significantly differed from the corresponding value of the MS12 strain according to the two-sample t-test; p < 0.05 and p < 0.01, respectively.