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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 Xa23SW11. 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.
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High‐efficiency prime editing enables new strategies for broad‐spectrum resistance to bacterial blight of rice
Summary Using genetic resistance against bacterial blight (BB) caused byXanthomonas oryzaepathovaroryzae(Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm againstXoo. Here, we use an improved prime‐editing system to implement two new strategies for BB resistance. Knock‐in of TAL effector binding elements (EBE) derived from the BB susceptible geneSWEET14into the promoter of a dysfunctional executorRgenexa23reaches 47.2% with desired edits including biallelic editing at 18% in T0generation that enables an inducible TALE‐dependent BB resistance. Editing the transcription factor TFIIA geneTFIIAγ5required for TAL effector‐dependent BB susceptibility recapitulates the resistance ofxa5at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0generation. The engineered loci provided resistance against multipleXoostrains in T1generation. Whole‐genome sequencing detected noOsMLH1dn‐associated random mutations and no off‐target editing demonstrating high specificity of this PE system. This is the first‐ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock‐in of 30‐nucleotides cis‐regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolvingXoostrains and protect it from epidemics.
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- Award ID(s):
- 1936492
- PAR ID:
- 10443056
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Plant Biotechnology Journal
- Volume:
- 21
- Issue:
- 7
- ISSN:
- 1467-7644
- Format(s):
- Medium: X Size: p. 1454-1464
- Size(s):
- p. 1454-1464
- Sponsoring Org:
- National Science Foundation
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