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.
Highlights
Cordyceps militaris is a kind of ascomycetous (Wang et al, 2008; Cui, 2015) farming mushroom with multiple medicinal (Reis et al, 2013) uses
With transformation with an single-guide RNA (sgRNA) that was synthesized in vitro, genome editing could be easy and flexible to apply in the traditional edible mushroom C. militaris
As a two-stage sac fungus, C. militaris is well-known for a high ratio of fruiting body gemmated degeneration in farming, which means a high dose of antifungal drugs might accelerate its degeneration to avoid the toxic effect
Summary
Cordyceps militaris is a kind of ascomycetous (Wang et al, 2008; Cui, 2015) farming mushroom with multiple medicinal (Reis et al, 2013) uses. Bioactive compounds, such as cordycepin (Tuli et al, 2015), ergosterol, ergothioneine (Cohen et al, 2014), cordyceps polysaccharides (Zhang et al, 2015), isolated from C. militaris have been demonstrated to have antitumorigenic (Wu et al, 2007), anti-inflammatory (Noh et al, 2008; Taofi et al, 2016), antioxidant (Kim et al, 2006) and antimicrobial functions (Zhou et al, 2016). Effective components in extracts could be useful materials in skin cosmetics (Chien et al, 2008) Due to these multiple applications, C. militaris has been regarded as a potential industrial mushroom in recent years. Traditional genomic editing technologies such as methods based on homologous recombination (HR) and Agrobacteriummediated random gene insertion were insufficient and too complicated to satisfy the requirement of accurate and repeatable editing
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