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Related Concept Videos

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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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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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CRISPRi-mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND)

Shih-I Tan1, Peng-Jui Yu1, I-Son Ng1

  • 1Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan.

Biotechnology and Bioengineering
|May 28, 2020
PubMed
Summary

We developed a novel Direct Enzymatic Performance Evaluation & Determination (DEPEND) system to rapidly screen enzyme performance. This method accelerates protein engineering by simplifying enzyme activity assessment.

Keywords:
CRISPRiaminolevulinic acid synthetasecarbonic anhydraseessential geneperformancewhole-cell biosensor

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Area of Science:

  • Biotechnology
  • Synthetic Biology
  • Enzyme Engineering

Background:

  • Enzyme screening is crucial for chemical production but is often time-consuming and labor-intensive.
  • Current methods require extensive manual labor and time for evaluating enzymatic activity.
  • Efficient screening is vital for advancing protein engineering and biocatalysis.

Purpose of the Study:

  • To develop a rapid, flexible, and efficient system for screening and evaluating enzyme performance.
  • To overcome the limitations of traditional, laborious enzyme screening processes.
  • To enable high-throughput assessment of enzymatic activity for protein engineering applications.

Main Methods:

  • Utilized a CRISPR interference (CRISPRi) system with a carbonic anhydrase (CA) gene (CRISPRi::CA) as a whole-cell biosensor for CO2 monitoring.
  • Developed the Direct Enzymatic Performance Evaluation & Determination (DEPEND) system through a single plasmid transformation step.
  • Applied the DEPEND system to assess carbonic anhydrase activity and 5-aminolevulinic acid synthetase (ALAS) performance, including chaperone effects.

Main Results:

  • Demonstrated a correlation between carbonic anhydrase activity and colony-forming units within the DEPEND system.
  • Successfully distinguished the enzymatic performance of different carbonic anhydrases.
  • Identified the effect of the GroELS chaperone on ALAS enzyme folding using the DEPEND system.

Conclusions:

  • The Direct Enzymatic Performance Evaluation & Determination (DEPEND) system offers a highly feasible, time-saving, and flexible technology for enzyme screening.
  • This novel system significantly accelerates the inspection and selection of high-performance enzymes.
  • The DEPEND system has the potential to greatly advance future protein engineering efforts.