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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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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.

Hana Raschmanová1, Astrid Weninger2, Karin Kovar3

  • 1University of Chemistry and Technology Prague, Department of Biotechnology, Prague, Czech Republic.

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|October 1, 2025
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Summary
This summary is machine-generated.

This study details a robust CRISPR/Cas9 genome editing protocol for Pichia pastoris, achieving nearly 100% efficiency for gene inactivation. This method facilitates genetic modifications and metabolic engineering in the yeast.

Keywords:
CRISPR/Cas9Gene knockoutGenome editingGenome engineeringGuide RNAHomologous recombinationKomagataella phaffiiPichia pastorisSynthetic biology

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Area of Science:

  • Molecular Biology
  • Yeast Genetics

Background:

  • The CRISPR/Cas9 system is a powerful tool for genome editing and metabolic engineering.
  • Established in 2016, CRISPR/Cas9 protocols for Komagataella phaffii (Pichia pastoris) enable genetic modifications like markerless gene disruptions.
  • Previous applications include enhancing homologous recombination efficiency.

Purpose of the Study:

  • To describe a robust, basic protocol for CRISPR-based genome editing in Pichia pastoris.
  • To demonstrate high targeting efficiency for gene inactivation using this protocol.

Main Methods:

  • Utilized the CRISPR/Cas9 system for genome editing in Pichia pastoris.
  • Employed a frameshift mutation strategy for gene inactivation.
  • Established a basic, robust protocol for P. pastoris genome editing.

Main Results:

  • Achieved near 100% targeting efficiency for gene inactivation.
  • Demonstrated the effectiveness of the CRISPR-based protocol for genetic modification.
  • The protocol is suitable for creating frameshift mutations for gene knockout.

Conclusions:

  • The described CRISPR/Cas9 protocol provides a reliable method for Pichia pastoris genome editing.
  • This protocol facilitates efficient gene inactivation and other genetic modifications.
  • Further optimizations of CRISPR/Cas9 technologies for P. pastoris are available for specific applications.