Open Access
International Journal of Molecular Sciences, volume 24, issue 9, pages 8173
A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts
Karginov Azamat V
1
,
Tarutina Marina G
2, 3
,
Lapteva Anastasia R.
2
,
Pakhomova Maria D.
1
,
Galliamov Artur A.
1
,
Filkin Sergey Y
1
,
Fedorov Alexey N
1
,
Publication type: Journal Article
Publication date: 2023-05-03
Quartile SCImago
Q1
Quartile WOS
Q1
Impact factor: 5.6
ISSN: 16616596, 14220067
Catalysis
Organic Chemistry
Inorganic Chemistry
Physical and Theoretical Chemistry
Computer Science Applications
Spectroscopy
Molecular Biology
General Medicine
Abstract
Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.
Citations by journals
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1
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Frontiers in Neuroscience
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Frontiers in Neuroscience
1 publication, 33.33%
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International Journal of Molecular Sciences
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International Journal of Molecular Sciences
1 publication, 33.33%
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Biomedicines
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Biomedicines
1 publication, 33.33%
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1
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Citations by publishers
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1
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Frontiers Media S.A.
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Frontiers Media S.A.
1 publication, 33.33%
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Multidisciplinary Digital Publishing Institute (MDPI)
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Multidisciplinary Digital Publishing Institute (MDPI)
1 publication, 33.33%
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1
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Karginov A. V. et al. A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts // International Journal of Molecular Sciences. 2023. Vol. 24. No. 9. p. 8173.
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Karginov A. V., Tarutina M. G., Lapteva A. R., Pakhomova M. D., Galliamov A. A., Filkin S. Y., Fedorov A. N., Agaphonov M. O. A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts // International Journal of Molecular Sciences. 2023. Vol. 24. No. 9. p. 8173.
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RIS
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TY - JOUR
DO - 10.3390/ijms24098173
UR - https://doi.org/10.3390%2Fijms24098173
TI - A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts
T2 - International Journal of Molecular Sciences
AU - Karginov, Azamat V
AU - Tarutina, Marina G
AU - Lapteva, Anastasia R.
AU - Pakhomova, Maria D.
AU - Galliamov, Artur A.
AU - Filkin, Sergey Y
AU - Fedorov, Alexey N
AU - Agaphonov, M. O.
PY - 2023
DA - 2023/05/03 00:00:00
PB - Multidisciplinary Digital Publishing Institute (MDPI)
SP - 8173
IS - 9
VL - 24
SN - 1661-6596
SN - 1422-0067
ER -
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@article{2023_Karginov,
author = {Azamat V Karginov and Marina G Tarutina and Anastasia R. Lapteva and Maria D. Pakhomova and Artur A. Galliamov and Sergey Y Filkin and Alexey N Fedorov and M. O. Agaphonov},
title = {A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts},
journal = {International Journal of Molecular Sciences},
year = {2023},
volume = {24},
publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},
month = {may},
url = {https://doi.org/10.3390%2Fijms24098173},
number = {9},
pages = {8173},
doi = {10.3390/ijms24098173}
}
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MLA
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Karginov, Azamat V., et al. “A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.” International Journal of Molecular Sciences, vol. 24, no. 9, May. 2023, p. 8173. https://doi.org/10.3390%2Fijms24098173.
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