期刊
NATURE PROTOCOLS
卷 15, 期 2, 页码 338-363出版社
NATURE PORTFOLIO
DOI: 10.1038/s41596-019-0238-3
关键词
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资金
- National Transgenic Science and Technology Program [2018ZX0801002B, 2019ZX08010001, 2018ZX0800102B, 2019ZX08010-003]
- National Natural Science Foundation of China [31788103, 31971370]
Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespread cis-elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR-Cas9 system. The method we present readily yields transgene-free uorf mutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)-Cas9 vectors, identifying transgene-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2-3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits. In this protocol, the authors describe a method to fine-tune gene expression in plants by editing endogenous upstream ORFs with the CRISPR-Cas9 system to prevent their inhibition of the translation of primary ORFs. This protocol yields transgene-free uorf mutant offspring.
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