Construction Of CRISPR-Cas9 Gene Editing System In Pleurotus Eryngi

Pleurotus eryngii,belonging to the Pleurotaceae,is a kind of precious edible fungus known as highly edible,nutritional,pharmaceutical,economic and ecological values.In view of the following features,including promoting digestion of the stomach,enhancing human immunity,lowering blood lipids,lowering cholesterol and preventing cardiovascular diseases,Pleurotus eryngii shall be favored among consumers and its annual output is getting higher and higher.However,fundamental research on molecular genetics of edible fungi is relatively weak and is far from meeting the growing demand for breeding and biotechnological applications without available molecular biology tools.Due to many restrictive factors for edible fungi,such as complex heredity law,amphidiploid as the existence form,it is fairly difficult for gene knockout and high-efficiency gene interference to successfully realize gene editing.Therefore,molecular genetics research is far from meeting the rapidly developing needs of edible fungi industry.At present,genome sequencing has been completed among a variety of edible fungi including Pleurotus eryngii.Taking the advantages of low cost,high accuracy of target recognition and easy operation into consideration,the new generation CRISPR-Cas9 gene editing technology has been successfully applied to animals,plants,and other kinds of food crops,which provides important data and technical support for the research on molecular genetics of edible fungi.The purpose of this study is to establish a stable and efficient system of genetic transformation and CRISPR-Cas9 gene editing system for Pleurotus eryngii.The research results are summarized as follows:1.The plasmid p EASY^?-Blunt-Pgpd-hph-trp C,containing the endogenous Pgpd promoter and hph–a positive selectable marker gene,was transformed into the protoplasts of Pleurotus eryngii by PEG-Ca Cl₂ mediated transformation of protoplasts.Acquisitions of transformants that successfully carried with hygromycin resistance contributed to the establishment of genetic transformation system using the hph gene as a resistance screening marker.2.Similarly,genetic transformation system was constructed using PEG-Ca Cl₂mediated transformation of protoplasts.Firstly,the full-length sequence of the sdh B of Pleurotus eryngii including promoter and terminator,enconding the iron-sulphurprotein subunit of succinate dehydrogenase,was cloned successfully.Then the comparison of Sdh B protein among three species including Pleurotus ostreatus,Coprinus cinereus and Pleurotus eryngii to find the 239th of conservative histidine site combined with the carboxin was conducted.Ultimately,that the histidine site was mutated to leucine successfully obtained the homologous screening marker gene defined as cbx^r for genetic transformation system of Pleurotus eryngii.Transformation of protoplasts along with cbx^rresulted in dominant resistence to carboxin,which indicated that site-mutated sdh B gene can be used as a dominant selective marker for transformation.Southern blot result revealed that the cbx^rgene conferring resistence to carboxin had been stably integrated in genome DNA of Pleurotus eryngii and appeared as a single copy in genome.3.According to the preference of genomic codons of Pleurotus eryngii,the open reading frame of Cas9 regarded as a core component of CRISPR-Cas9 system was optimized maximumly.After that,the protein expression and nuclear localization signal of Cas9 were both confirmed.Meanwhile,the promoter of Pol III RNA polymerase that drives sg RNA transcription was excavated in the genome of Pleurotus eryngii in efforts to screen out four functional U6 sn RNA promoters named as PEU6-1.PEU6-2、PEU6-3 and PEU6-4.Subsequent protoplast transformation tests verified that the transcription activity of sg RNA was stronger than that of the T7 promoter in vitro.4.The pyr G gene was used as an example to analyze the types and genetic characteristics of mutations induced by the optimized CRISPR-Cas9 system.Research results showed that genetic variations mediated by the CRISPR-Cas9 system involving indels–insertion and deletion–of Non-homologous end joining（NHEJ）,as well as the replacement of GFP fragments of homologous recombination（HR）.Genetic variation could be stably transmitted to the next generation and CRISPR-Cas9 system integrated into Pleurotus eryngii cells could still be active to edit the modified target genes.On the basis of uracil auxotrophic strain of Pleurotus eryngii that losed the function of glycoside-5’-phosphate decarboxylase,synonymous point-mutated pyr G gene at designated seven bases position was re-intergrated to restore normal growth phenotype,which proved that synonymous point-mutated pyr G gene might be used as a screening marker to construct a genetic transformation system when using uracil auxotrophic strain of Pleurotus eryngii as background.This study demonstrated the construction of a genetic transformation system inedible mushroom Pleurotus eryngii and the CRISPR-Cas9 gene editing system,by which above methods could be widely applied not only to diverse mushroom species other than Pleurotus eryngii,but also to breed improvement for mushroom,regulation in metabolic pathway,boost in functional biology and research on molecular genetics.