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

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 AOX2* ‐KpA3A‐nCas9‐KpUGI‐ DAS1 TT) comprised a truncated AOX2 promoter (P AOX2* ), 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 ‐18 to C ‐12 . 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 .