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

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 ad...

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Bibliographic Details
Published inBiotechnology journal Vol. 19; no. 7; p. e2400115
Main Authors Wu, Ling-Yu, Xu, Yan, Yu, Xiao-Wei
Format Journal Article
LanguageEnglish
Published Germany 01.07.2024
Subjects
CRISPR-Cas Systems - genetics
Cytidine Deaminase - genetics
Cytidine Deaminase - metabolism
Gene Editing - methods
Humans
Promoter Regions, Genetic - genetics
Proteins
Saccharomycetales - genetics
CRISPR
Komagataella phaffii
nSpRy
nCas9
base editing
APOBEC3A
nSpG
Online AccessGet more information
ISSN1860-7314
DOI10.1002/biot.202400115

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Summary: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 C to 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.
ISSN:1860-7314
DOI:10.1002/biot.202400115