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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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...
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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
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A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.

Xihao Liao1,2, Lu Li1,2, Aysha Jameel1,2

  • 1MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China.

Applied Microbiology and Biotechnology
|November 13, 2021
PubMed
Summary

We developed a CRISPR-based toolkit for Pichia pastoris, enabling efficient gene editing and transcriptional regulation. This system accelerates applications in metabolic engineering and synthetic biology for microbial cell factories.

Keywords:
CRISPR/Cas9Combinatorial genome engineeringPichia pastorisTranscriptional regulation

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Area of Science:

  • Microbial biotechnology
  • Synthetic biology
  • Molecular genetics

Background:

  • Pichia pastoris is a key microbial cell factory for producing proteins and chemicals.
  • Limited genome engineering tools hinder Pichia pastoris applications in metabolic engineering and synthetic biology.

Purpose of the Study:

  • To develop a versatile CRISPR-based toolbox for gene editing and transcriptional regulation in Pichia pastoris.
  • To enhance Pichia pastoris capabilities for advanced metabolic engineering and synthetic biology.

Main Methods:

  • Constructed a CRISPR/Cas9 system for gene editing with RNA Pol-III-driven sgRNA expression and an eliminable episomal plasmid.
  • Developed CRISPR/dCas9 tools fused with transcriptional repressors (Mix1/RD1152) or activators (VPR) for gene expression regulation.
  • Engineered a CRISPR-ARE system for simultaneous gene activation, repression, and editing using multiplex sgRNAs.

Main Results:

  • Achieved up to 100% gene knockout efficiency using the CRISPR/Cas9 system.
  • Demonstrated strong gene repression (above 70%) and activation (up to 3.5-fold) of reporter genes.
  • Showcased the CRISPR-ARE system's efficiency in individual and combinatorial gene activation, repression, and editing.

Conclusions:

  • The developed CRISPR toolbox provides simple and multifunctional tools for Pichia pastoris.
  • This toolkit will significantly accelerate Pichia pastoris applications in metabolic engineering and synthetic biology.
  • The system enables precise genetic manipulation for optimizing microbial cell factories.