CRISPR-Cas9 Assisted Gene Disruption In The Higher Fungus Ganoderma Lucidum

Mushrooms,a large class of higher fungi,have many edible and medicinal values,which can produce many useful natural products which have important biological functions,such as reducing blood pressure,enhancing immunity,and possessing anti-cancer and anti-HIV and other pharmacological activities.Therefore,mushrooms have attracted intense attention by academia and industry.However,the lack of mature genetic manipulation method,especially gene disruption,not only restricted the understanding of the metabolic regulation of mushrooms,but also hindered further strain modification.Previous gene disruptions in mushrooms were mainly based on homologous recombination(HR),while their HR efficiencies were extremely low,for example,the number of transformants with a gene deletion was only one or two when transformed with 10~7 protoplasts of Schizphylhls commne.Therefore,there is no report about gene disruption in many important medicinal mushrooms,such as Ganoderma lucidum,Poria cocos,Lentinus edodes,etc.Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR-associated protein 9(CRISPR-Cas9)is one of the most famous gene editing tools.With high efficiency and easy operation,CRISPR-Cas9 system has recently been applied to many species,but there has no report in mushrooms.By adopting CRISPR-Cas9 from Streptococcus pyogenes,gene disruption was developed in G.lucidum in this study.Using codon-optimized Cas9 and in vitro transcribed gRNA,ura3 gene of G.lucidum 260125 and G.lucidum 5.616 were successfully disrupted.At the same time,we found that the addition of gRNA can increase the efficiency of gene disruption,and two mutants could be obtained from 10~7 protoplast.In order to interrupt other functional genes though CRISPR-Cas9 technology,homologous recombination mediated by CRISPR has been explored in G.lucidum.In order to promote the efficiency of homologous recombination,the promoters of U6 small nuclear RNA(U6snRNA)which could drive gRNA endogenous transcription in Ganoderma have been found by sequence alignment.To our knowledge,this is the first example of using CRISPR-Cas9 system for gene disruption in mushrooms,which provides useful platform for basic and applied research in mushrooms.