Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.A...Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.展开更多
基金supported by the National Natural Science Founda-tion of China(U23A20268)the National Natural Science Foundation of Shandong Province(ZR2022ZD24)the Taishan Scholar Project of Shandong Province(tsqn202312061).
文摘Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.
