Genome editing has offered a powerful tool for crop breeding, and how to modulate endogenous protein levels has become crucial for creating new elite alleles. Recent studies revealed that the ATG start codon immediate upstream 5′-UTR sequence (AUS) is an important cis-control feature in human cells and Arabidopsis protoplasts. Especially, the occurrence of a trinucleotide AAA in the AUS correlates with a high translation efficiency in Arabidopsis protoplasts, while a trinucleotide TTT is associated with decreased translation efficiency. We expected the prime editing (PE) system could be applied to design the AUS to create artificial alleles of target genes without affecting coding regions in rice. In this study, we chose two genes, a strigolactone biosynthesis gene OsHTD1 (high tillering and dwarf 1) and a gibberellin metabolism gene OsSBI (shortened basal internodes) to examine whether the rational design of their AUSs could create useful alleles to acquire Green Revolution traits. We first tested the protein output of the target genes' AUS in rice protoplasts via a dual-luciferase reporter assay. Then, we applied the PE technology to introduce the AAA insertion at OsSBI AUS and the TTT insertion at OsHTD1 AUS as designed in protoplasts. The results showed that OsSBI protein abundance was strongly elevated in OsSBI^aus lines compared to that of WT, consistently, the plant heights of OsSBI^aus lines were significantly reduced. However, OsHTD1 protein abundance was largely decreased in OsHTD1^aus lines and their tiller numbers were significantly increased compared to WT. These results suggest that the rational design of AUS in OsSBI and OsHTD1 could rapidly create beneficial alleles to obtain Green Revolution traits in rice, providing a convenient and practical tool in crop breeding.

Genome editing has offered a powerful tool for crop breeding, and how to modulate endogenous protein levels has become crucial for creating new elite alleles. Recent studies revealed that the ATG start codon immediate upstream 5′-UTR sequence (AUS) is an important cis-control feature in human cells and Arabidopsis protoplasts. Especially, the occurrence of a trinucleotide AAA in the AUS correlates with a high translation efficiency in Arabidopsis protoplasts, while a trinucleotide TTT is associated with decreased translation efficiency. We expected the prime editing (PE) system could be applied to design the AUS to create artificial alleles of target genes without affecting coding regions in rice. In this study, we chose two genes, a strigolactone biosynthesis gene OsHTD1 (high tillering and dwarf 1) and a gibberellin metabolism gene OsSBI (shortened basal internodes) to examine whether the rational design of their AUSs could create useful alleles to acquire Green Revolution traits. We first tested the protein output of the target genes' AUS in rice protoplasts via a dual-luciferase reporter assay. Then, we applied the PE technology to introduce the AAA insertion at OsSBI AUS and the TTT insertion at OsHTD1 AUS as designed in protoplasts. The results showed that OsSBI protein abundance was strongly elevated in OsSBI^aus lines compared to that of WT, consistently, the plant heights of OsSBI^aus lines were significantly reduced. However, OsHTD1 protein abundance was largely decreased in OsHTD1^aus lines and their tiller numbers were significantly increased compared to WT. These results suggest that the rational design of AUS in OsSBI and OsHTD1 could rapidly create beneficial alleles to obtain Green Revolution traits in rice, providing a convenient and practical tool in crop breeding.

Genome editing has offered a powerful tool for crop breeding, and how to modulate endogenous protein levels has become crucial for creating new elite alleles. Recent studies revealed that the ATG start codon immediate upstream 5′-UTR sequence (AUS) is an important cis-control feature in human cells and Arabidopsis protoplasts. Especially, the occurrence of a trinucleotide AAA in the AUS correlates with a high translation efficiency in Arabidopsis protoplasts, while a trinucleotide TTT is associated with decreased translation efficiency. We expected the prime editing (PE) system could be applied to design the AUS to create artificial alleles of target genes without affecting coding regions in rice. In this study, we chose two genes, a strigolactone biosynthesis gene OsHTD1 (high tillering and dwarf 1) and a gibberellin metabolism gene OsSBI (shortened basal internodes) to examine whether the rational design of their AUSs could create useful alleles to acquire Green Revolution traits. We first tested the protein output of the target genes' AUS in rice protoplasts via a dual-luciferase reporter assay. Then, we applied the PE technology to introduce the AAA insertion at OsSBI AUS and the TTT insertion at OsHTD1 AUS as designed in protoplasts. The results showed that OsSBI protein abundance was strongly elevated in OsSBI^aus lines compared to that of WT, consistently, the plant heights of OsSBI^aus lines were significantly reduced. However, OsHTD1 protein abundance was largely decreased in OsHTD1^aus lines and their tiller numbers were significantly increased compared to WT. These results suggest that the rational design of AUS in OsSBI and OsHTD1 could rapidly create beneficial alleles to obtain Green Revolution traits in rice, providing a convenient and practical tool in crop breeding.