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CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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Efficient CRISPR-mediated C-to-T base editing in Komagataella phaffii
Ling-Yu Wu1, Yan Xu1, Xiao-Wei Yu1
1Lab of Brewing Microbiology and Applied Enzymology, School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi, China.
Biotechnology Journal
|July 10, 2024
Summary
Researchers developed a novel CRISPR-nCas9 base editing tool for Komagataella phaffii. This tool enables efficient C-to-T base editing, advancing genetic engineering in this important yeast species.
Area of Science:
- Biotechnology
- Synthetic Biology
- Microbial Engineering
Background:
- Komagataella phaffii is a methylotrophic yeast utilized for producing industrial enzymes, pharmaceuticals, and chemicals.
- Advanced genome editing tools are essential for developing K. phaffii into more sophisticated cell factories.
Purpose of the Study:
- To develop a robust and versatile base editing method for Komagataella phaffii.
- To engineer and optimize CRISPR-nCas9 based base editors for efficient C-to-T base editing in K. phaffii.
Main Methods:
- Engineered 24 base editor constructs using various promoters and cytidine deaminases (CDAs).
- Optimized a base editor (PAOX2*-KpA3A-nCas9-KpUGI-DAS1TT) for K. phaffii.
- Tested base editors with modified nCas9 variants (nSpG, nSpRy) to expand targetable genomic regions.
Main Results:
- The optimal base editor achieved high C-to-T editing efficiencies: 96.0% single-locus, 65.0% double-locus, and 5.0% triple-locus editing.
- Editing occurred within a specific window (C-18 to C-12).
- Modified nCas9 variants enabled editing at NGN and NRN protospacer adjacent motif (PAM) sites with efficiencies ranging from 20.0% to 93.2%.
Conclusions:
- Developed powerful base editing tools for K. phaffii.
- These tools facilitate gene function research, metabolic engineering, and genetic improvement in K. phaffii.
- The expanded targeting capabilities enhance functional genomics research in this yeast.
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