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Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
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Cloning and validating systems for high throughput molecular recording.

Anqi Zhao1, Michelle M Chan1

  • 1Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, United States; Department of Molecular Biology, Princeton University, Princeton, NJ, United States.

Methods in Enzymology
|March 22, 2025
PubMed
Summary
This summary is machine-generated.

This study details a CRISPR-Cas9 based molecular recorder for high-resolution lineage tracing. The optimized system enables precise tracking of cellular history during development and disease.

Keywords:
CRISPR-Cas9Cellular engineeringGenome editingLineage tracingMolecular recorder

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Molecular recording technologies capture cellular history.
  • Lineage tracing is crucial for understanding development, stem cell maintenance, and tumor evolution.
  • Existing methods may lack the resolution needed for complex biological processes.

Purpose of the Study:

  • To describe optimized procedures for constructing a CRISPR-Cas9 based molecular recorder system for lineage tracing.
  • To validate the performance of this novel technology for high-resolution cellular history recording.
  • To provide guidance on technology design, cloning, cell line generation, and experimental assessment.

Main Methods:

  • Utilizing CRISPR-Cas9 barcode editing to introduce mutations in engineered DNA cassettes.
  • Genomic integration of DNA cassettes for stable recording.
  • Readout via single-cell RNA sequencing.
  • Development of optimized cloning and validation protocols.

Main Results:

  • Successful construction of a molecular recorder lineage tracing system.
  • Demonstration of high-resolution lineage tree reconstruction.
  • Validation of optimized cloning and cell line generation procedures.
  • Assessment of system performance through time course experiments.

Conclusions:

  • The described molecular recorder technology offers a powerful tool for high-resolution lineage tracing.
  • Optimized procedures facilitate the construction and application of this system.
  • This technology has broad implications for studying mammalian development, stem cell biology, and cancer evolution.