A simple and universal system for gene manipulation in Aspergillus fumigatus

In vitroassembled Cas9-guide RNA ribonucleoproteins coupled with microhomology repair templates

Qusai Al Abdallah, Wenbo Ge, Jarrod Fortwendel

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 50 bp 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 2 μg of the repair template flanked by homology regions as short as 35 bp. 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, 2 μg of the repair template flanked by 35 and 50 bp 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.

Original languageEnglish (US)
Article numbere00446-17
JournalmSphere
Volume2
Issue number6
DOIs
StatePublished - Nov 1 2017

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Guide RNA
Ribonucleoproteins
Aspergillus fumigatus
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Deletion
Genes
Technology
Homologous Recombination
DNA

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

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title = "A simple and universal system for gene manipulation in Aspergillus fumigatus: In vitroassembled Cas9-guide RNA ribonucleoproteins coupled with microhomology repair templates",
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 50 bp 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 2 μg of the repair template flanked by homology regions as short as 35 bp. 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, 2 μg of the repair template flanked by 35 and 50 bp 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.",
author = "{Al Abdallah}, Qusai and Wenbo Ge and Jarrod Fortwendel",
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AU - Fortwendel, Jarrod

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