ArticleCell reports methods2026
Parallel gene amplification by Cas9 nickase for generating functionally heterogeneous cell populations.
Article in Cell reports methods, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Genetic diversity underlies adaptive evolution. Because genes often act in concert to execute biological processes, the dosage stoichiometry among cooperating genes represents an additional layer of diversity beyond sequence variation. We therefore hypothesized that combinatorial randomization of gene copy numbers could generate cell populations enriched for functional heterogeneity and evolutionary potential. To test this idea, we extended our previously developed Cas9 nickase-based gene amplification method, break-induced replication-mediated tandem repeat expansion (BITREx), to simultaneously target multiple genes. Applying parallel BITREx to three carotenogenic genes introduced into the budding yeast Saccharomyces cerevisiae, we generated a cell population exhibiting broad variation in both absolute copy numbers and their stoichiometric ratios. This population enabled the identification of elite genotypes-specific copy number combinations that conferred enhanced β-carotene production. These results suggest that parallel BITREx is a versatile strategy for increasing functional heterogeneity in cell populations, with potential applications in both basic and applied research.
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