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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

252
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
252

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Related Experiment Video

Updated: Sep 13, 2025

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Development of precise genome editing and multi-copy integration tools in Hansenula polymorpha DL-1.

Xiaoyi Zou1,2, Jiaqi Miao1,2, Hongbiao Li1,2

  • 1Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.

Synthetic and Systems Biotechnology
|July 30, 2025
PubMed
Summary

This study developed advanced synthetic biology tools for Hansenula polymorpha yeast, improving genome editing and gene integration for sustainable manufacturing of valuable compounds.

Keywords:
CRISPR-Cas9Hansenula polymorpha DL-1Homologous recombinationMulti-copy integrationNeutral sites

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Area of Science:

  • Microbial biotechnology
  • Synthetic biology
  • Yeast genetics

Background:

  • Hansenula polymorpha DL-1 is a thermotolerant yeast with potential for sustainable manufacturing.
  • Its utility is limited by strong non-homologous end joining (NHEJ) and few genetic tools.

Purpose of the Study:

  • To develop an optimized synthetic biology toolkit for H. polymorpha DL-1.
  • To enhance genome editing efficiency and homologous recombination (HR) rates.
  • To enable stable single-copy and multi-copy gene integration for increased production.

Main Methods:

  • Established a high-efficiency CRISPR-Cas9 genome editing system.
  • Suppressed NHEJ by knocking out KU80 and overexpressing S. cerevisiae HR genes.
  • Identified 36 neutral sites for single-copy integration.
  • Developed multi-copy integration tools targeting rDNA and Ty elements.

Main Results:

  • Achieved 97.2% editing efficiency with CRISPR-Cas9.
  • Increased HR rates to 88.9% by modifying NHEJ and HR pathways.
  • Enabled stable single-copy integration without disrupting native genes.
  • Achieved a ~60-fold increase in beta-carotene and enhanced squalene production to 187.2 mg/L via multi-copy integration.

Conclusions:

  • The developed toolkit significantly enhances the genetic tractability of H. polymorpha DL-1.
  • This facilitates its use as a versatile platform for efficient sustainable production of value-added compounds.