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Exploiting the efficient Exo:Cas12i3-5M fusions for robust single and multiplex gene editing in rice

文献类型： 外文期刊

作者： Wang, Wenxue 1 ; Li, Shaoya 1 ; Yang, Jiaying 1 ; Li, Jingying 1 ; Yan, Lei 1 ; Zhang, Chen 1 ; He, Yubing 1 ; Xia, Lanqin 1 ;

作者机构： 1.Chinese Acad Agr Sci CAAS, Inst Crop Sci ICS, State Key Lab Crop Gene Resources & Breeding, Beijing 100081, Peoples R China

2.Chinese Acad Agr Sci, Natl Nanfan Res Inst Sanya, Key Lab Gene Editing Technol Hainan, Minist Agr & Rural Affairs, Sanya 572024, Peoples R China

关键词： Cas12i3-5M; exonucleases; multiplex gene editing; rice (Oryza sativa L.)

期刊名称：JOURNAL OF INTEGRATIVE PLANT BIOLOGY （ 影响因子：9.3； 五年影响因子：10.8 ）

ISSN： 1672-9072

年卷期： 2025 年 67 卷 5 期

页码：

收录情况： SCI

摘要： The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif (PAM). However, due to its relatively lower editing efficiency, Cas12i3-mediated multiplex gene editing has not yet been documented in plants. Here, we fused four 5 ' exonucleases (Exo) including T5E, UL12, PapE, ME15 to the N terminal of an optimized Cas12i3 variant (Cas12i3-5M), respectively, and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines. We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M, respectively. Notably, the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61% across the six tested endogenous genes. We further demonstrated that, although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing, UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three, four, five or six genes with efficiencies of 82.76%, 61.36%, 52.94%, and 51.06% in rice stable lines, respectively. Together, we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice. The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.

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