| Summary: | Abstract Background The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. Results The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. Conclusions Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.
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