CAPE System: Revolutionizing Plant Breeding with Promoter Editing

In plant functional genomics research, the acquisition of target gene function loss mutant editing materials is a key factor and effective way to interpret gene function. Similarly, the creation of specific gene promoter mutant editing materials is also a necessary verification system for plant promoter site confirmation, element identification, and functional interpretation. However, existing plant promoter research mainly relies on the identification of natural promoter element variant materials, large sample GWAS screening, EMS and irradiation mutagenesis materials. Although such work has achieved important results in the identification and application of plant promoter-related functions, it inevitably has defects such as long cycle, large investment, strong randomness, and poor reliability.

With the rapid expansion of the breadth and depth of genome editing technology in basic research on plant gene function and molecular breeding practice, effective editing of plant gene promoters, obtaining promoter editing materials with changed expression levels or expression patterns of target genes, and then effectively interpreting the biological functions of target promoters and creating new promoter editing germplasms with breeding value will inevitably become a hot research field. However, the current editing tools based on Cas9 have obvious technical blind spots in editing non-coding sequences such as promoters. Researchers are in urgent need of efficient promoter editing tools and feasible editing strategies that can significantly improve the efficiency of plant promoter editing, the polymorphism of editing events, and the yield of "non-silent editing events".

On April 6, 2023, Zhang Yong from the University of Electronic Science and Technology of China and his collaborators published a research paper entitled "An efficient CRISPR-Cas12a promoter editing (CAPE) system for crop improvement" in Nature Plants, developing an efficient CRISPR-Cas12a promoter editing (CAPE) system based on a prediction model of multi-omics data. Based on the CAPE system, the authors accurately predicted the key regulatory sites in the promoter region of plant target genes, designed high-confidence site guide RNAs, and constructed multi-site or single-site CRISPR-Cas12a editing expression vectors (libraries). They achieved linear adjustment of target gene expression levels and corresponding agronomic traits in rice OsGBSS1 and OsGS3, and then mined and identified new alleles (OsO18-PEs) based on promoter editing strategies, providing an effective strategy for de novo creation of crop quantitative traits.

Based on the DNA sequence and structural characteristics of plant promoter regions, combined with the internal factors of cells that effectively regulate promoter transcription, researchers have integrated multi-omics data such as open chromatin, transcription factor binding sites, histone modifications, genomic polymorphisms, and transcriptomes to develop an intelligent prediction algorithm for key regions and sites of plant promoters based on a multi-factor weighted assignment strategy. Combined with the characteristics of Cas12a and the diverse needs of plant promoter editing, an efficient plant CAPE system has been constructed that can realize the credibility evaluation of key editing regions or sites of plant genome-wide promoters, the selection of editing tools, and the optimization of editing sites. It also provides an online genome browser tool.

Figure 1. Design of CAPE system. (Zhou, et al., 2023)

Based on the CAPE strategy, the authors achieved high-confidence prediction of key regions and sites of target gene promoters for rice endogenous genes such as OsGBSS1 and OsGS3. Based on the auxiliary design of guide RNA and editing tool selection suggestions, they completed effective promoter editing work for the OsGBSS1 and OsGS3 genes, significantly improving the overall yield of promoter editing events and the genotypic polymorphism of editing events. Among them, the yield of effective editing materials that change the target traits has been significantly improved, and a series of promoter editing materials with complete gene structures and continuous changes in transcription levels of OsGBSS1 and OsGS3 have been obtained. The author identified a number of promoter editing materials with practical breeding value, confirming the feasibility of de novo creation of quantitative traits based on the CAPE system.

The CAPE system was further used to edit the promoters of key genes for the biosynthesis of hormones such as gibberellins, brassinolides and strigolactones in rice. It was found that the plant height of the Nipponbare OsD18 (OsGA3ox2) gene promoter-edited materials showed continuous changes, and the important agronomic traits of the recipient materials were maintained. In further experiments, the OsD18 gene promoter was edited in the two varieties YBN2 and BBN1, and high-yield semi-dwarf materials were also obtained. Field trials showed that the OsD18 promoter-edited series of materials of Nipponbare, YBN2 and BBN1 with different genetic backgrounds all showed the same agronomic trait performance and lodging resistance phenotype as the OsSD1 mutant.

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Reference

1. Zhou,J., et al. An efficient CRISPR-Cas12a promoter editing system for crop improvement. Nat Plants. 2023, 9(4): 588-604.