Establishment Of A Dual SgRNA-directed Gene Deletion Method By CRISPR/Cas9 System In Ganoderma Lingzhi

Ganoderma lingzhi(G.lingzhi),an important group of higher medicinal fungi,can produce various kinds of natural products,such as ganoderic acid,polysaccharide,and alkaloid.They have been proved to have anti-cancer,anti-oxidation,anti-HIV-1,and other important biological activities.With the study of G.lingzhi,the genome and transcriptome sequencing of G.lingzhi have been completed.Although its genome sequence was obtained,there is little information available on functions for those genes in the genome.Therefore,investigation of those gene functions is significant for understanding of the growth,development and metabolic regulation of G.lingzhi.At present,the methods for studying on the function of G.lingzhi genes mainly include gene overexpression and gene silencing.However,further functional characterization of some genes has been limited due to the lack of an effective gene deletion system in G.lingzhi.Traditionally,the gene deletion technique is mainly based on homologous recombination(HR)with low efficiencies(0.1-1%)in higher fungi.The CRISPR/Cas9 system has been used as the basis for developing a simple and efficient tool for genome editing in many organisms.For example,disruption of some genes in higher fungi such as Cordyceps militaris,Pleurotus ostreatus and G.lingzhi have been achieved recently.Nevertheless,there has been no report of gene deletions via the CRISPR/Cas9 system in G.lingzhi.To express effectively the Cas9 gene in G.lingzhi,the heterologous gene expression system of G.lingzhi was firstly established in this study.Effects of codon optimization and intron addition on the expression of hygromycin resistance gene were evaluated in G.lingzhi.Our results showed that hygromycin-resistant transformants can be generated by adopting codon optimization and intron addition simultaneously in G.lingzhi.Moreover,we found that this method is also applicable to successfully express other heterologous genes such as the glufosinate resistance gene and green fluorescent protein gene.Based the established method,the engineering strain efficient expressing the Cas9 gene(p JW-EXP-intron-opcas9)was obatained for gene disruption in G.lingzhi.Here,we described targeted gene disruption in p JW-EXP-intron-opcas9 engineering strain via the CRISPR/Cas9 system.The sgRNA1 and sgRNA2 were designed to target the orotidine monophosphate decarboxylase gene(ura3)of G.lingzhi.In vitro transcribed sgRNA1 were adopted for transformation into the p JW-EXP-intron-opcas9 engineering strain and the codon-optimized traditional Cas9(Glcas9)strain,respectively.Results showed the destruction efficiency of the ura3 in the the p JW-EXP-intron-opcas9 was increased by 10.6 times compared with that in the glcas9 engineering strain.For sgRNA1,the Sanger sequencing results demonstrated that the destruction efficiency was as high as 100% in all detected 30 transformants.Among those transformants,25±2(83.3±6.6%)of the transformants showed insertion mutants,4±1(13.3±3.3%)of the transformants showed deletion mutants,and 1±0(3.3±0%)of the transformants showed replacement mutant.For sgRNA2,the destruction efficiency was as high as 100% in 20 transformants.But insertion,deletion,and replacement efficiency were 65±5%,35±5%,and 0%,repectively.Our results demonstrated that both sgRNA1 and sgRNA2 are effective in gene disruption of ura3.The disruption efficiency was different,perhaps because that the Cas9-generated alleles are sequence-dependent biases.Then,we established a targeted gene deletion method in G.lingzhi via a dual sgRNA directed CRISPR/Cas9 system.In vitro transcribed sgRNA1 and sgRNA2 were mixed and transformed into protoploasts of G.lingzhi(p JW-EXP-intron-opcas9)at a concentration of 100 ?g.The mutation efficiency of the CRISPR/Cas9 system for each sgRNA was also determined by Sanger sequencing.Destruction efficiencies of 28.5±4%,and 34.6±2% for sgRNA1,and sgRNA2 were obtained in 49 transformant.18±3 transformants(the deletion efficiency was 36.7± 6%)were found to be gene deletion of the target sites of sgRNA1 and sgRNA2.Our results illustrated the CRISPR/Cas9 system is an efficient tool for gene deletion in G.lingzhi.In summary,we found that codon optimization and intron addition are important for heterologous gene expression in G.lingzhi.Moreove,we established a targeted gene deletion method in G.lingzhi via a dual sgRNA directed CRISPR/Cas9 system.This may also contribute to the functional study of genomics and molecular breeding in G.lingzhi in the future.