CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar

Abstract

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Keywords: 5-FOA resistant mutation; Gene-specific mutagenesis; Mucormycosis pathogen; PyrF and PyrG.

Fig. 1.. Characterization of spontaneous pyrF- mutants of Rhizopus delemar FGSC-9543 and CDC-8219 strains.

(A) Typical appearance of spontaneous 5-FOA resistant colonies of CDC-8219 after incubation for 5 days at 30 °C. The typical pyrF- mutant is pointed by a white arrow. The colony pointed by the black arrow typically failed to retain 5-FOA resistance in subsequent culturing. (B) DNA sequencing of the 3.0-kb fragment amplified by primers PW1819 and PW1827 that contains the pyrF 5′-UTR and ORF. (C) Growth of the R. delemar pyrF- f2 mutant in comparison to wild type CDC-8219 and complemented TF1 and TF3 strains on potato dextrose agar (PDA), MMC –uracil, and MMC + 5-FOA (1 g/L). Incubation was carried out at 30°C for 1–5 days as indicated. (D) DNA sequencing of the 217-bp PCR fragment amplified with primers PW1972 and PW1945 (top chromatograms) and cloned products (lower chromatograms). (E) Primers PW2015 and PW2050 were used for PCR (30 cycles for the pyrF-dpl200 control and 35 cycles for the rest) to analyze complemented pyrF- mutant strains. A ~400-bp is expected for the pyrF- mutant and ~600-bp is expected for the integrated pyrF-dpl200 marker. Lane 1: pyrF-dpl200, lane 2: pyrF(91–92ΔTC), lane 3 and 4: complemented TF #1 and TF #3, respectively.

Fig. 2.. Diagram of pmCas9:tRNA-gRNA for CRISPR-Cas9 in R. delemar.

Schematic representation of partial pmCas9-tRNA-gRNAF1 plasmid containing Cas9 and gRNA expression cassettes. A Ustilaginoidea virens Gln-tRNA sequence exhibiting Pol III promoter activity (indicated in dark blue ink was used to drive the expression of gRNA. There are two inverted BsmBI/Esp3I restriction enzyme sites (in black ink) built into the sequence following the Gln-tRNA promoter. This construct is not cleavable by BsmBI/Esp3I once the annealed 20-nt gRNA plus 4 nt linker sequence (pyrF1 is illustrated in light blue ink) is inserted.

Fig. 3.. Occurrence of 5-FOA resistant colonies following CRISPR-Cas9 mediated electroporation.

Representative plates following transformation with pmCas9:tRNA-gRNA. pmCas9:tRNA-pyrF1 and pmCas9:tRNA-pyrF2 were electroporated into 50 μl of swollen spores with the setting of time constant, 1100 V, and 5 ms. Spores were allowed to recover for 90 min before plating onto the selective MMC medium. Plates were incubated for 5–7 days or until colonies emerged. The pyrF::HYG fragment (~3-kb) obtained by PCR amplification with primers PW1823 and PW1827 was used as homologous donor DNA. Arrows point to the putative pyrF-transformants.

Fig. 4.. CRISPR-Cas9 induces single point deletion in pmCas9-tRNA-pyrF1 target site of the pyrF gene.

Sequencing chromatograms and corresponding sequence diagrams of CRISPR-Cas9 induced pyrF- mutations in FGSC-9543 and CDC-8219 strains. Primer PW1972 and PW2050 were used to amplify the pyrF- PCR fragments that were sequenced directly and sequenced after cloning into pCR2.1. The protospacer adjacent motif (PAM) sequences were shaded in light grey in both chromatograms and sequence diagrams. Arrows point to the position of single nucleotide deletions.