Nature Protocols
Abstract
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.
| 论文编号: | DOI:10.1038/s41596-019-0238-3 |
| 论文题目: | Manipulating Gene Translation in Plants by CRISPR–Cas9-mediated Genome Editing of Upstream Open Reading Frames |
| 英文论文题目: | Manipulating Gene Translation in Plants by CRISPR–Cas9-mediated Genome Editing of Upstream Open Reading Frames |
| 第一作者: | Xiaomin Si, Huawei Zhang, Yanpeng Wang, Kunling Chen, Caixia Gao |
| 英文第一作者: | Xiaomin Si, Huawei Zhang, Yanpeng Wang, Kunling Chen, Caixia Gao |
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| 发表年度: | 2020-01-09 |
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| 摘要: | 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. |
| 英文摘要: | 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. |
| 刊物名称: | Nature Protocols |
| 英文刊物名称: | Nature Protocols |
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| 其它备注: | Xiaomin Si, Huawei Zhang, Yanpeng Wang, Kunling Chen, Caixia Gao. Manipulating Gene Translation in Plants by CRISPR–Cas9-mediated Genome Editing of Upstream Open Reading Frames. Nature Protocols. DOI:10.1038/s41596-019-0238-3 |
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