Journal: Fungal genetics and biology : FG & B
Article Title: CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar
Figure Lengend Snippet: 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.
Article Snippet: A 200-bp fragment from the 5′ UTR of the R. delemar pyrF gene was tandemly incorporated into the downstream, generating the pyrF-dpl200 marker gene ( ).
Techniques: Incubation, Mutagenesis, DNA Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Marker