TY - JOUR
T1 - Efficient Generation of Multiple Seamless Point Mutations Conferring Triazole Resistance in Aspergillus fumigatus
AU - Handelman, Mariana
AU - Osherov, Nir
N1 - Publisher Copyright:
© 2023 by the authors.
PY - 2023/6
Y1 - 2023/6
N2 - Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus.
AB - Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus.
KW - ARMS-PCR
KW - Aspergillus fumigatus
KW - CRISPR-Cas9
KW - genetic manipulation
KW - pTel
UR - http://www.scopus.com/inward/record.url?scp=85163761359&partnerID=8YFLogxK
U2 - 10.3390/jof9060644
DO - 10.3390/jof9060644
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C2 - 37367580
AN - SCOPUS:85163761359
SN - 2309-608X
VL - 9
JO - Journal of Fungi
JF - Journal of Fungi
IS - 6
M1 - 644
ER -