Abstract :
[en] CRISPR/Cas-mediated genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. However, most of the reports mainly focus on targeted mutagenesis for gene knock-out and base editing for single base transition. Precise targeted gene/allele replacement or gene tagging in plants still remains very challenging, especially in crop plants. Here, we briefly summarized the so far reported strategies for precision genome editing in plants. Based on the recently developed prime editing strategy which enables gene replacement without double-strand breaks (DSBs) or donor repair templates (DRT) in mammalian cells, we further propose the future perspectives of exploiting diverse strategies to achieve efficient precise targeted gene/allele replacement to accelerate crop improvement and resilience for sustainable agricultural development. An opinion on precision genome editing in crop plants is presented in Chapter Ⅱ.
Prime editing enables precise installation or replacement of short indels in a specific gene without requiring double-strand DNA breaks and donor repair templates in human cells. This novel genome-editing technology offers the potential to improve precision editing efficiency in crop plants. However, it remains unclear whether a similar strategy will work efficiently in plant species. In this study, we developed a primer editor system to edit both an exogenous mutated hptII gene and an endogenous OsEPSPS gene in rice. We successfully achieved both homozygous and heterozygous stable lines with desired precise modifications in these two genes. The precision efficiencies reached at 9.38% and 2.22% for mutated hptII and OsEPSPS, respectively. Thus, we have established an efficient prime editing system, providing a feasible and effective tool for the precision editing of the rice genome. The detail of the results is presented in Chapter Ⅳ.
The ability to manipulate multiple genomic sites in a predefined manner has great significance in pyramiding beneficial alleles in one variety for crop improvement. So far, only accurate modification of a single gene by prime editing has been reported in different species including crop plants at variable efficiencies. Multiplex precise gene editing by prime editing has not been documented yet. In this study, we developed three surrogate prime editors including hygromycinY46*-based, OsALSS627I-based, and a combined double surrogate system, respectively, for prime editing of endogenous genes with substantially improved editing efficiency in rice stable lines. While the hygromycinY46*-based and OsALSS627I-based surrogate prime editors could increase the editing efficiencies by ~2-14-fold, the double surrogate system could stimulate the prime editing efficiencies up to ~50-fold. Furthermore, we precisely edited several endogenous genes simultaneously and obtained stable lines by using this double surrogate system. Together, our surrogate prime editors demonstrated substantially improved editing efficiency and enabled multiplex precise gene editing, thereby greatly expanding the potential of prime editing in the concurrent improvement of multiple traits in crop plants. The detail of the results is presented in Chapter V.
Aphid is one of the most devastating agricultural important pests which causes significant yield losses each year. Aphid is also a model species in dissecting plant-insect interactions. This is further accentuated by the fact that nearly all currently commercialized crop cultivars lack endogenous aphid-resistance genes and are susceptible to aphid infestation. Over the past decade, advanced technologies including RNA interference (RNAi) and genome editing technologies have been extensively applied in the functional analysis of genes in various organisms including aphids and plants, respectively. In Chapter I, we summarize the latest progress in the applications of RNAi and genome editing technologies in dissecting plant-aphid interactions and generating aphid-resistant plants. We also propose future perspectives on applications of these technologies in aphid biological research, plant-aphid interaction, as well as the generation of novel crop germplasm resistant to aphid infestation. In this work, we identify aphid resistance-related genes (DSR48) from grain aphids. Transgenic RNAi lines were obtained based on plant-mediated RNAi technology, and the potential function of DSR48 in plant-aphid interactions will be dissected. The details of the results are presented in chapter VI.
