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Related Experiment Video

Updated: May 27, 2026

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
09:04

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

Parallel gene amplification by Cas9 nickase for generating functionally heterogeneous cell populations.

Hiroaki Takesue1, Satoshi Okada1, Takashi Ito1

  • 1Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan.

Cell Reports Methods
|May 25, 2026
PubMed
Summary

Researchers explored genetic diversity by randomizing gene copy numbers. This approach created varied cell populations, leading to the discovery of gene combinations that boost beta-carotene production in yeast.

Keywords:
BITRExCP: biotechnologyCP: systems biologybreak-induced replicationgenome editingpathwayyeast

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Last Updated: May 27, 2026

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Area of Science:

  • Synthetic biology
  • Metabolic engineering
  • Evolutionary biology

Background:

  • Genetic diversity is crucial for adaptive evolution.
  • Gene dosage and stoichiometric ratios offer a layer of diversity beyond sequence variation.
  • Combinatorial randomization of gene copy numbers can enhance functional heterogeneity and evolutionary potential.

Purpose of the Study:

  • To test the hypothesis that randomizing gene copy numbers can generate functionally heterogeneous cell populations.
  • To extend the break-induced replication-mediated tandem repeat expansion (BITREx) method for simultaneous multi-gene targeting.
  • To identify specific gene copy number combinations that enhance biological product formation.

Main Methods:

  • Developed and applied parallel BITREx for simultaneous gene amplification.
  • Targeted three carotenogenic genes in Saccharomyces cerevisiae.
  • Generated a yeast population with diverse absolute copy numbers and stoichiometric ratios of target genes.

Main Results:

  • Successfully created a cell population with broad variation in gene copy numbers and ratios.
  • Identified elite genotypes with specific copy number combinations for enhanced beta-carotene production.
  • Demonstrated the effectiveness of parallel BITREx in generating functional heterogeneity.

Conclusions:

  • Parallel BITREx is a versatile strategy for increasing functional heterogeneity in cell populations.
  • This method has potential applications in both basic research and biotechnology.
  • Randomizing gene copy numbers is a viable approach to uncover beneficial genetic combinations for desired traits.