Introduction:
Aspergillus
fumigatus is a species of fungus in the genus Aspergillus and is one
of the most common Aspergillus species to cause disease in individuals with
immunodeficiency. It can be found throughout the environment, including in
soil, plant matter, and household dust. Humans and animals constantly inhale
numerous conidia of this fungus. The conidia are normally eliminated in the
immunocompetent host by innate immune mechanisms, and aspergilloma and allergic
bronchopulmonary aspergillosis, uncommon clinical syndromes. Thus, A. fumigatus
was considered for years to be a weak pathogen. Nowadays,
Aspergillus fumigatus becomes more and more popular in research because this specie plays an important role in the environment and human life.
Also, CRISPR/Cas9 is a novel genome-editing
system that has been successfully established in Aspergillus fumigatus. Scientists
have generated a CRISPR system for Aspergillus fumigatus to study its function
by gene knockout, gene
knockin, point mutation, etc.
Aspergillus fumigatus is one of the most common causes of aspergillosis. The first one is allergic bronchopulmonary aspergillosis, it is an allergic reaction to the Aspergillus spores. This reaction can lead to damage in your airways and lungs. It is often found in people that have conditions such as asthma and cystic fibrosis. The second one is Chronic pulmonary aspergillosis, which can occur in people with chronic lung conditions that cause air spaces called cavities to form in the lung. The third one is invasive aspergillosis, it is the most severe form of aspergillosis and can be fatal if not treated. It occurs when an aspergillosis infection begins in the lungs and spreads to other parts of the human body, such as skin, brain, or kidney. Invasive aspergillosis occurs only in people who have a severely weakened immune system.
Development of the CRISPR/Cas9 System for Targeted
Gene Disruption in Aspergillus fumigatus
To test the CRISPR/Cas9 system’s feasibility
for targeted gene disruption in A. fumigatus. As a proof of principle, researchers
first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25
to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as
evidenced by the generation of colorless (albino) mutants harboring the
expected genomic alteration. Researchers further demonstrated that the
constitutive expression of the Cas9 nuclease by itself is not deleterious to A.
fumigatus growth or virulence, thus making the CRISPR system compatible with
studies involved in pathogenesis. Taken together, these data demonstrate that
CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the
potential to bolster the genetic toolbox for this important pathogen.
Using CRISPR to Gene Manipulation in Aspergillus
fumigatus
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 technology is limited to strains
that are engineered to express Cas9 and gRNA. To overcome such limitations, researchers
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, the CRISPR-Cas9 system utilizes 35 to 50 bp of flanking
regions for mediating homologous recombination at Cas9 double-strand breaks
(DSBs). Similar deletion efficiencies were obtained in the clinical isolate
DI15-102. The data shows that in vitro-assembled Cas9 RNPs coupled with
microhomology repair templates are an efficient and universal system for gene
manipulation in A. fumigatus. In this study, 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. The simple and universal CRISPR-Cas9 system for gene
manipulation in this study generates efficient gene targeting across different
genetic backgrounds of A. fumigatus.
Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing
vectors and processes. The efficiency and accuracy are much higher than
traditional methods. CRISPR-B™ can be used in gene editing of bacteria and
fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and
knockin (KI).
References:
Latgé JP. Aspergillus fumigatus and
aspergillosis. Clin Microbiol Rev. 1999;12(2):310-350
Development of the CRISPR/Cas9 System for
Targeted Gene Disruption in Aspergillus fumigatus
Kevin K. Fuller, Shan Chen, Jennifer J.
Loros, Jay C. Dunlap
Eukaryotic Cell Oct 2015, 14 (11) 1073-1080
A Simple and Universal System for Gene
Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA
Ribonucleoproteins Coupled with Microhomology Repair Templates
Qusai Al Abdallah, Wenbo Ge, Jarrod R.
Fortwendel
mSphere Nov 2017, 2 (6) e00446-17; DOI:
10.1128/mSphere.00446-17
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