Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes in Aspergillus fumigatus

• DOI: 10.1186/s40694-018-0057-2
• Publication Date: 2018-04-27T11:17:51Z
• Authors: Qusai Al Abdallah; Ana Camila Olivei...; Adela Martin-Vicente; Wenbo Ge; Jarrod R. Fortwendel
• Keywords: 0301 basic medicine; 03 medical and health sciences; Aspergillus fumigatus; Off-target mutation; Short Report; CRISPR/Cas9; TP248.13-248.65; Genome editing; Biotechnology

CRISPR/Cas9-based genome editing is quickly becoming a powerful tool within the field of fungal genetics. Adaptation CRISPR/Cas9 systems are allowing for rapid and highly efficient gene targeting fungi. We recently reported adaptation simple system deletion that effective across multiple genetic backgrounds Aspergillus fumigatus. This employs in vitro assembly Cas9 ribonucleoproteins (RNPs) coupled with micro-homology repair templates deletion. Although at wild type A. fumigatus, potential our to produce unwanted off-target mutations has not been addressed.Next-generation Illumina sequencing was used identify among transformants isolated from standard (no Cas9) Cas9-mediated integration hygromycin cassette. Two different concentrations were utilized examine association concentration total numbers types genomic mutations. For each three test groups (zero, low, high Cas9), sequenced compared parent strain. Bioinformatics analyses revealed average number be similar all groups. fumigatus transformation using standard, non-Cas9-mediated methods resulted an 373 ± 28 In comparison, assembled Cas9-RNPs either (1 µg/µl) or low (0.5 levels 326 19 395 69 mutations, respectively. cases, vast majority identified intergenic. No correlation between amount overall found. Finally, specific mutation introduced during process Cas9-dependent, as both single-nucleotide polymorphisms insertion/deletion events significantly experimental groups.CRISPR/Cas9-based RNPs microhomology reliable method targeting. associated increased caused by introduction nuclease.