Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris

Abstract

Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.

Keywords: CRISPR-Cas9; Cordyceps militaris; edible-medicinal mushroom; fungi industry; gene disruption.

Figure 1

Flow diagram of CRISPR-Cas9 vector construction. (A) Pcmgpd, Pcmlsm3 and Tcmura3 were cloned from the genome of C. militaris. (B) The cassette Pcmgpd-cmcas9-gfp-myc was ligated into pCAMBIA0390 to build the p390cm-gpd-c9gm vector. (C) The cassette Pcmlsm3-blpR-Tcmura3 was ligated into p390cm-gpd-c9gm to build p390-blpR-cmcas9-gfp.

Figure 2

The qPCR experiments were carried out in biological triplicate. Data are represented as the mean ± SEM (n = 9). Statistical analyses were performed using t-tests compared with wild-type “CM10” (^***p < 0.001, ^****p < 0.0001). (A) The expression level of sgRNA in the transformant C. militaris C9-sR (C. militaris:: blpR-cmcas9-gfp-HH-sgRNA-HDV). (B) The expression level of Cas9 in the transformant C. militaris C9 (C. militaris:: blpR-cmcas9-gfp).

Figure 3

Detection of Cas9-expressing transformants. (A) Nucleic acid gel electrophoresis analysis of putative Cas9 transformants (Expected target band was 491 bp). (B) Western blot for Cas9 detection (S1–S5 represented putative transformants; CM10 represented the negative control; the expected Cas9 band was 186.5 kDa and is indicated by arrows).

Figure 4

Mycelia of the Cas9-GFP transformant C9 and negative control CM10 were shown under (A) bright field image and (B) fluorescence microscope (excitation, 395 to 440 nm, and emission, 470 nm). The bars were 20 μm.

Figure 5

Sequence design and alignment diagrams for editing cmura3 by CRISPR-Cas9 in vivo. (A) The localized sites and target sequences of sgRNA. (Apostrophe in the box represents the sgRNA scaffold sequence). (B) The sequence of donor ssDNA ortUra3-1. (The target sgRNA site was underlined; the inserted sequence was marked blue and listed separately). (C) The sequence and sequencing chromatogram for gUra3-1 deletion mutation. (D) The sequences and sequencing chromatograms for gUra3-1 replacement mutation. (E) The sequences and sequencing chromatograms for gUra3-1 insertion mutation and the alignments for the insertions. (F) The sequence and sequencing chromatogram for the gUra3-2 inserted mutation and alignment for the insertions.