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

Abstract

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