Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Ajay Gupta; Bo Liu; Saad Raza; Qi-Jun Chen; Bing Yang

doi:10.1016/j.xplc.2023.100741

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Plant Commun. 2024 Feb 12;5(2):100741. doi: 10.1016/j.xplc.2023.100741. Epub 2023 Oct 26.

Authors

Ajay Gupta¹, Bo Liu¹, Saad Raza¹, Qi-Jun Chen², Bing Yang³

Affiliations

¹ Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
² State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, China.
³ Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA; Donald Danforth Plant Science Center, St. Louis, MO 63132, USA. Electronic address: yangbi@missouri.edu.

Abstract

Prime editing (PE) technology enables precise alterations in the genetic code of a genome of interest. PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants, ideally targeting multiple loci simultaneously to realize the collective effects of the edits. Here, we report the development of a modular assembly-based multiplex PE system in rice and demonstrate its efficacy in editing up to four genes in a single transformation experiment. The duplex PE (DPE) system achieved a co-editing efficiency of 46.1% in the T₀ generation, converting TFIIAγ5 to xa5 and xa23 to Xa23^SW11. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae (Xoo) strains in the T₁ generation. In addition, we successfully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele, achieving a co-editing rate of 57.14%. Furthermore, with the quadruple PE (QPE) system, we edited four genes-two for herbicide tolerance (OsEPSPS1 and OsALS1) and two for Xoo resistance (TFIIAγ5 and OsSWEET11a)-using one construct, with a co-editing efficiency of 43.5% for all four genes in the T₀ generation. We performed multiplex PE using five more constructs, including two for triplex PE (TPE) and three for QPE, each targeting a different set of genes. The editing rates were dependent on the activity of pegRNA and/or ngRNA. For instance, optimization of ngRNA increased the PE rates for one of the targets (OsSPL13) from 0% to 30% but did not improve editing at another target (OsGS2). Overall, our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents, making it feasible for more labs to utilize PE for their editing experiments. These findings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

Keywords: bacterial blight; herbicide tolerance; multiplex genome editing; prime editing; rice.

MeSH terms

Alleles
Gene Editing
Herbicides* / pharmacology
Oryza* / genetics

Substances

Herbicides