Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology

Abstract A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii ). Plasmids containing single guide RNA and the methanol expression regulator 1 ( MXR1 ) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single‐base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris . The expression levels of several of the Mxr1‐targeted genes, AOX1 , AOX2 , DAS1 , and DAS2 , in strains containing the various mutated variants of MXR1 , were then detected through reverse transcription PCR following induction in methanol‐containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1 , DAS1 , and DAS2 , while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1 , AOX2 , DAS1 , and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1 , DAS1 , and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.