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

Abstract

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

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

“…oryzae and A . fumigatus genome editing with Cas9-RNP did not seem to induce off-target effects [ 24 , 48 ], and did not increase the mutation frequency of transformants compared to standard mutagenesis procedures [ 48 ]. To evaluate the occurrence of Cas9-RNP-mediated off-site integrations and the overall frequency of transformation-associated off-target mutations in B .…”

“…Even if sgRNA sequences are chosen that avoid highly similar secondary regions in the genome, off-site integrations have been observed in sites with several mismatches [47]. Nevertheless, in studies performed with M. oryzae and A. fumigatus genome editing with Cas9-RNP did not seem to induce off-target effects [24,48], and did not increase the mutation frequency of transformants compared to standard mutagenesis procedures [48]. To evaluate the occurrence of Cas9-RNPmediated off-site integrations and the overall frequency of transformation-associated off-target mutations in B. cinerea, genome sequencing was performed with the following strains: B05.10 (WT reference), a mutant generated by standard targeted mutagenesis (nep2-NatR), a different amino acids in sdhB codon 272.…”

Progress and Challenges: Development and Implementation of CRISPR/Cas9 Technology in Filamentous Fungi

“…After transformation of the fungus with either a high concentration of Cas9 RNPs (1 μg/μL) or a low concentration (0.5 μg/μL), whole genome sequencing revealed there was no significant difference in the number of mutations in either one of the Cas9 RNP mediated transformations when compared to traditional transformation [77]. Additionally, there was no difference in the types of mutations that occurred between these groups, as the number of insertions and deletions were similar, and the locations of the mutations were predominantly located in intergenic regions [77]. In another study, off-target mutations were evaluated after CRISPR/Cas9-mediated transformation directed to TRI5 in F. graminearum [60].…”

Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms

“…The combination of these markers with the use of photo-convertible/switchable tags and more-powerful fluorescence microscopy techniques such as super-resolution microscopy (adapted to A. nidulans by the groups of N. Takeshita and R. Fischer) will significantly improve our understanding of the localization/dynamics/ reorganization of protein complexes (Perez-de-Nanclares-Arregi and Etxebeste 2014; Etxebeste and Takeshita 2015;Ishitsuka et al 2015). Although site-directed mutagenesis procedures in A. nidulans are highly efficient and easily reproducible, the adaptation of CRISPR/Cas9 technology is a must, as has been already done for other Aspergilli (Fuller et al 2015;Nødvig et al 2015;Zhang et al 2016;Al Abdallah et al 2018). Similarly, the study of protein complexes and the elucidation of their structure also require improved proteomic procedures, such as the one developed for the isolation of NPCs of S. cerevisiae and the determination of the type/ amount of each NUP or the morphology of the entire pore (Kim et al 2018).…”