Background
Different gene expressions can lead to different plant phenotypes. Therefore, establishing a method for fine regulation of genes based on gene editing technology is crucial for precise breeding design. Currently, the most widely used gene expression regulation methods, such as CRISPR-Cas, CRISPRi, and RNAi, can only achieve complete gene knockout or suppress gene expression at unpredictable levels.
Editing the promoter region can regulate gene expression at the transcriptional level, resulting in quantitative phenotypic changes for breeding purposes. However, screening ideal mutants using this method is time-consuming and labor-intensive. Therefore, developing new strategies for finely regulating gene expression can significantly expand the existing gene expression regulation toolbox and provide technical support for crop genetic improvement.
Upstream open reading frames (uORFs) are short protein-coding elements located in the 5′ untranslated regions (UTRs) of primary open reading frames (pORFs), and the presence of uORFs is associated with reduced mRNA translation. Many factors, such as the length of the uORF and the distance between the uORF and the pORF, can affect the inhibitory ability of the uORF. However, there are few approaches to finely downregulate the expression of endogenous genes at the translational level in plants.
Experiment and Result
Generation of Novel uORFs to Repress Protein Expression
In this study, the researchers inhibited the translation of the target gene through the following strategy. They generate a new uORF at the 5' UTR of the target gene. The researchers used base editing and prime editing to create short uORFs in the 5'UTRs of coding genes to achieve quantitative downregulation of endogenous gene expression. Analysis of the 5'UTRs of genes can identify those genes without uORFs in their 5'UTRs. Then, by mutating or inserting 1-3 bases to generate upstream ATG start codons, several de novo uORFs can be generated upstream of these genes.
Prolonging Endogenous uORFs Reduces Protein Expression
For genes with their own uORFs, researchers extend the original uORF or generate additional uORF by site-directed mutation of the stop codon of the endogenous uORF to extend the length of its expression frame.
Results using a transient reporter system, such as a dual-luciferase assay, demonstrated that these strategies could effectively repress the translation of pORF to varying levels with little effect on its mRNA expression. A plasmid containing the 35S promoter is provided by GenScript to achieve dual luciferase detection.
In addition, by detecting the protein expression level and phenotype of the mutant rice plants obtained by the above strategies, it was found that the effect of the uORF introduced in the mutant on the expression and phenotype of the target protein was consistent with the results of the transient system.
Gradient Downregulation of Protein Expression
Based on the above strategies, the researchers believe that generating uORFs with different inhibitory activities in the 5’ UTR of genes may be able to control the degree of down-regulation of gene expressions. Therefore, the researchers selected OsTB1, OsTCP19, and OsDLT genes, and combined the above strategies to design a series of uORFs with different inhibitory abilities in the 5' UTR region of the genes. These uORFs gradually downregulated the translation of pORFs to 2.5-84.9% of wild-type levels, achieving a gradient knockdown of genes.
It is worth mentioning that the OsDLT gene encodes a member of the GRAS family and participates in the brassinosteroid transduction pathway, regulating multiple critical agronomic traits such as plant height, tiller number, and seed size in rice. By editing the 5'UTR of the OsDLT gene, a series of mutants with different plant heights and tiller numbers were obtained, and the phenotypic change trend was consistent with the prediction results of the transient system.
Conclusion
In summary, the study of precise genome editing to edit uORFs of 5′UTRs of plant-encoding genes has developed a general and predictable new method for finely down-regulating gene expression, which provides a necessary technical means for future genetic design of crop breeding.
Reference
Xue, C., Qiu, F., Wang, Y. et al. Tuning plant phenotypes by precise, graded downregulation of gene expression. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01707-w
