Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast , a broader variety of employable...

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Bibliographic Details
Published in:Journal of fungi (Basel) 2024-03, Vol.10 (3), p.197
Main Authors: Smirnov, Kirill, Weiss, Florian, Hatzl, Anna-Maria, Rieder, Lukas, Olesen, Kjeld, Jensen, Sanne, Glieder, Anton
Format: Article
Language:English
Subjects:
CRISPR
CRISPR-Cas9
E coli
Efficiency
Genes
genome editing
Genomes
Glycerol
Glycerol kinase
Intellectual property
Kinases
Komagataella phaffii
Localization
MAD7 nuclease
Nuclease
Pichia pastoris
Plasmids
Proteins
R&D
Research & development
RNA polymerase
Yeast
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Summary:CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast , a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 ( ), red fluorescence protein ( ), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in . In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).
ISSN:2309-608X
2309-608X
DOI:10.3390/jof10030197