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Efficient CRISPR-mediated C-to-T base editing in Komagataella phaffii.
- Source :
-
Biotechnology journal [Biotechnol J] 2024 Jul; Vol. 19 (7), pp. e2400115. - Publication Year :
- 2024
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Abstract
- The nonconventional methylotrophic yeast Komagataella phaffii is widely applied in the production of industrial enzymes, pharmaceutical proteins, and various high-value chemicals. The development of robust and versatile genome editing tools for K. phaffii is crucial for the design of increasingly advanced cell factories. Here, we first developed a base editing method for K. phaffii based on the CRISPR-nCas9 system. We engineered 24 different base editor constructs, using a variety of promoters and cytidine deaminases (CDAs). The optimal base editor (P <subscript>AOX2*</subscript> -KpA3A-nCas9-KpUGI-DAS1TT) comprised a truncated AOX2 promoter (P <subscript>AOX2*</subscript> ), a K. phaffii codon-optimized human APOBEC3A CDA (KpA3A), human codon-optimized nCas9 (D10A), and a K. phaffii codon-optimized uracil glycosylase inhibitor (KpUGI). This optimal base editor efficiently performed C-to-T editing in K. phaffii, with single-, double-, and triple-locus editing efficiencies of up to 96.0%, 65.0%, and 5.0%, respectively, within a 7-nucleotide window from C <subscript>-18</subscript> to C <subscript>-12</subscript> . To expand the targetable genomic region, we also replaced nCas9 in the optimal base editor with nSpG and nSpRy, and achieved 50.0%-60.0% C-to-T editing efficiency for NGN-protospacer adjacent motif (PAM) sites and 20.0%-93.2% C-to-T editing efficiency for NRN-PAM sites, respectively. Therefore, these constructed base editors have emerged as powerful tools for gene function research, metabolic engineering, genetic improvement, and functional genomics research in K. phaffii.<br /> (© 2024 Wiley‐VCH GmbH.)
Details
- Language :
- English
- ISSN :
- 1860-7314
- Volume :
- 19
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Biotechnology journal
- Publication Type :
- Academic Journal
- Accession number :
- 38987223
- Full Text :
- https://doi.org/10.1002/biot.202400115