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CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
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CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

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Logical regulation of endogenous gene expression using programmable, multi-input processing CRISPR guide RNAs.

Hansol Kang1, Dongwon Park1, Jongmin Kim1

  • 1Department of Life Sciences, Pohang University of Science and Technology, Pohang 37673, Korea.

Nucleic Acids Research
|June 29, 2024
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Summary
This summary is machine-generated.

Engineered conditional guide RNAs (gRNAs) offer precise control over CRISPR-Cas systems in E. coli. This breakthrough enables dynamic gene regulation for metabolic engineering and cell morphology studies.

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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Microbiology

Background:

  • CRISPR-Cas systems offer versatile RNA-guided applications.
  • Engineering guide RNAs (gRNAs) allows conditional control of CRISPR-Cas.
  • Precise regulation of CRISPR-Cas for metabolic processes is challenging.

Purpose of the Study:

  • Develop a robust dCas9 regulator with engineered conditional gRNAs.
  • Enable tight control of endogenous genes using conditional gRNAs.
  • Demonstrate conditional gRNA applications in Escherichia coli.

Main Methods:

  • Engineered conditional gRNAs interacting with trigger RNAs for gene expression control.
  • Utilized a dCas9 regulator system in Escherichia coli.
  • Evaluated gene expression dynamic range and logic gate operations (A OR (B AND C)).
  • Targeted endogenous metabolic genes (lacZ, malT, poxB) and cytoskeleton genes (ftsZ, mreB).

Main Results:

  • Conditional gRNAs achieved up to 130-fold dynamic range in gene expression.
  • Demonstrated control of metabolic flux and growth via targeting metabolic genes.
  • Regulated cell filamentation and division by targeting cytoskeleton genes.
  • Implemented two-input logic gates for conditional ftsZ regulation, inducing morphological changes.

Conclusions:

  • Engineered conditional gRNAs provide precise, programmable control over gene expression in E. coli.
  • The system enables modulation of cellular processes including metabolism and morphology.
  • This platform is potentially compatible with other Cas-effectors and host organisms.