Research On The Application Of CRISPR-Cas9 Gene Editing System In The Ghost Umbrella

Coprinopsis cinerea is commonly used in studying development of the developing fruiting body of basidiomycete.The functions of thousands of genes still remain unknown thoμgh whole genome sequencing of Coprinopsis cinerea has finished.The gene knockout by homologous recombination is not feasible because of its low efficency.CRISPR-Cas9 is a system that a noncoding RNA guides Cas9 protein to cut genome near the recognition sequence,and produces double-stranded DNA(DSBs).DSBs stimglate cells homologous recombination repair or non-homologous end joining(NHEJ)repair,so as to realize the target of foreign genes inserted or orientation of endogenous gene knock out.In this experiment,we consider Coprinopsis cinerea as the object of research,and we want to establish a CRISPR-Cas9 system to study the functions of important genes of Coprinopsis Cinerea.In the fungal transformation system,the cell wall of fungal cell is treated with enzymatic hydrolysis to prepare protoplasts,it is a commonly used methods to prepare recipient cells.ChiⅢ and ChiEn1 were heterologously expressed in Pichia pastoris,who play an important role in autolysis of the fruiting body of basidiomycete coprinoid.They were used to replace commercial chitinase to enzymolyse cell walls in transformation system of Coprinopsis cinerea.The experiment resμlts show that chitinase ChiⅢ with the same enzyme activity coμld replace commercial chitinase to enzymolyse cell walls and participate in forming protoplasts,and cooperative action occurs when administered together with ChiEn1.Our laboratory has been using nutritional deficient strain AmutBmut to screen positive posterity while transforming into Coprinopsis cinerea.Because of lacking efficient selection marker,it is impossible to knock out two genes or mμltiple genes simμltaneously.For making better use of CRISPR-Cas9 system,we use carboxin as a selection marker.Its gene seguence origins from Coprinopsis cinerea so it is esay to express with low concentration and low cost.using the carboxin resistant selection marker is the premise that establish CRISPR-Cas9 system.We try to establish CRISPR-Cas9 system in Coprinopsis cinerea,and we want to deliver plasmids that encode Cas9 protein and sgRNA to Coprinopsis cinerea,but this method works on condition that promoters of cas9 protein and sgRNA coμld be found.However,the U6 promoter is undefined and cas9 gene is not expressed in Coprinopsis cinerea.On this occasion,delivery of preassembled Cas9 protein-gRNA ribonucleoproteins woμld be a better choice.The experiment resμlts show that Cas9 protein doesn’t cleave target gene in vitro and in vivo,this may be related to easy degradation of Cas9 protein and sgRNA or low activity of designed sgRNA.We can improve CRISPR-Cas9 system by designing more sgRNAs,improving the experiment environment without RNA enzyme or optimizing cas9 gene sequence.