Precised Genome Editing Of Filamentous Fungi Based On TALENs

Aspergillus niger is widely found in nature,commonly in soil,food and plant products.As the superior in protein expressing and secreting,A.niger was widely applied on the production of various types of metabolites,such as citric acid,cellulose,penicillin,etc.In order to increase the industrial producted capacity and to reduce unnecessary products,genome-editting is the key to reach the goal in genetically.As a protein originally found in plant pathogenic bacteria,transcription activator-like effector(TALEs)can fuse with the cleaving domain of restriction endonuclease(for example Fok I)to form artificial nucleases named TALENs.These proteins are dependent on variable numbers of tandem repeats of TALEs to recognize and bind DNA sequences.Each of these repeats consists of a set of approximately 34 amino acids,composed of about 32 conserved amino acids and 2 highly vaciable amino acids called Repeat Variant Di-residues(RVDs).RVDs distinguish one TALE from another and can make TALEs have a simple cipher for the one-to-one recognition for proteins and DNA bases.Based on this,in theory,artificially constructed TALENs could recognize and break DNA sites specifically and arbitrarily to perform gene knockout,insertion or modification.In recent years,TALENs become a new gene editing technology,which specifically recognizes and cleaves DNA domain to achieve the purpose of editing the specified gene.In this study,the TALENs of A.niger was designed to target the gene GlaA(Accession number:AM270061).Two pairs of TALEN targeting vector was successfully constructed.A method of PEG-mediated transformation of protoplast was attempted;and the electrotransformation targeted to A.niger was successfully constructed by the expression vector pANEP8-hygro;ATMT method was also done well in A.niger.