Abstract
CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A.fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuBku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2µg of the repair template flanked by homology regions as short as 35bp. Next, we inspected the portability of our system across other genetic backgrounds of A.fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A.fumigatus DI15-102. In the Af293 strain, 2µg of the repair template flanked by 35 and 50bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A.fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A.fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A.fumigatus. We anticipate that our system will simplify genome editing in A.fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A.fumigatus.
Highlights
CRISPR-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus
Our goal was to establish a simple and universal system for complete gene deletion that coupled in vitro assembly of dual Cas9 RNPs with a repair template that is flanked by microhomology regions adjacent to the target gene
PksP mediates the biosynthesis of dihydroxynaphthalene (DHN)-melanin, a secondary metabolite that accounts for the gray-green color of conidia in A. fumigatus [29]
Summary
CRISPR (clustered regularly interspaced short palindromic repeat)-Cas is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas is an emerging tool for programmable genome editing in prokaryotes and eukaryotes In this technique, which was originally discovered in bacteria as a mechanism of acquired resistance against viral infections, the Cas DNA nuclease recognizes and cleaves specific DNA sequences after forming a ribonucleoprotein (RNP) complex with a guide RNA (gRNA) [3,4,5]. The HDR pathway integrates an endogenous sister chromatid or an exogenous repair template containing large regions of DNA homologous to sequences flanking the DSB site. With the widespread use of CRISPR-Cas for genome editing, the MMEJ pathway has been employed for Cas9mediated manipulations by using exogenous repair templates containing microhomology regions flanking the DSB site [11]. Whereas HDR requires long homology flanking regions (~500 to 5,000 bp) for precise insertion of the repair template, MMEJ-based integration of the repair template is mediated by short microhomology regions (2 to 40 bp) [12, 17,18,19]
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