title: Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus.
abstract: Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Meng
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Wei
authors.affiliation: Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China. Electronic address: linglu@njnu.edu.cn.
authors.forename: Ling
authors.initials: L
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000235:genetics*; D064113:CRISPR-Cas Systems; D003062:Codon; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics
chemical_list: D003062:Codon; D005656:Fungal Proteins; D012320:RNA Polymerase III
keywords: CRISPR mutagenesis; In situ tag-insertion; Microhomology; Multi-locus genomic mutagenesis