Efficient CRISPR‐mediated C‐to‐T base editing in Komagataella phaffii

Jul 10, 2024Biotechnology journal

Accurate CRISPR-based DNA editing in Komagataella phaffii

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Abstract

The optimal base editor for Komagataella phaffii achieved C-to-T editing efficiencies of up to 96.0% for single-locus modifications.

Twenty-four different base editor constructs were engineered using various promoters and cytidine deaminases.
The optimal base editor combines a truncated AOX2 promoter, a human APOBEC3A CDA, human codon-optimized nCas9, and a uracil glycosylase inhibitor.
Editing efficiencies for double- and triple-locus modifications were observed at 65.0% and 5.0%, respectively.
Replacing nCas9 with nSpG and nSpRy expanded the targetable genomic region, achieving 50.0%-60.0% and 20.0%-93.2% editing efficiencies for different PAM sites.
These base editors may serve as effective tools for gene function research and metabolic engineering in K. phaffii.

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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-KpA3A-nCas9-KpUGI-DAS1TT) comprised a truncated AOX2 promoter (P), 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 Cto C. 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. AOX2 AOX2 -18 -12

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