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| Main Authors: | , , , , , , , , , , , , |
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| Format: | Artículo científico |
| Language: | en |
| Published: |
Nature communications
2026
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| Online Access: | https://pubmed.ncbi.nlm.nih.gov/42277013/ |
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Table of Contents:
- High-throughput Raman-activated cell sorting of microalgal genome-wide edited library revealed a regulatory pathway for carotenoid synthesis. Wang, Qintao Gong, Yanhai Wang, Lianhong Lv, Nana Du, Xuefeng Zhang, Jiashun Xin, Yi Nikoloski, Zoran Li-Beisson, Yonghua Sun, Luyang Ma, Bo Wang, Xixian Xu, Jian Functional genomics have been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated cell sorting (RACS). The CRISPR/Cas-generated library consisted of 3567 microalgal mutants derived from 2397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unraveled mutations in the violaxanthin de-epoxidase (noVDE) or in the proteasome assembly chaperone 4 (noPAC4) genes. Knocking out all five known noVDEs revealed that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidence suggested two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this forward-genetics approach can greatly accelerate the discovery of genes and pathways.