Wang Wenxue, Li Shaoya, Yang Jiaying, Li Jingying, Yan Lei, Zhang Chen, He Yubing, Xia Lanqin
State Key Laboratory of Crop Gene Resources and Breeding, Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100081, China.
Key Laboratory of Gene Editing Technologies (Hainan), Ministry of Agricultural and Rural Affairs /National Nanfan Research Institute (Sanya), Chinese Academy of Agricultural Sciences, Sanya, 572024, China.
J Integr Plant Biol. 2025 May;67(5):1246-1253. doi: 10.1111/jipb.13850. Epub 2025 Jan 28.
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.
对于功能基因组学或作物改良中有益等位基因的聚合而言，开发单一和多重基因编辑系统非常必要。CRISPR/Cas12i3属于II类V-I型Cas系统，由于其蛋白质尺寸较小且对典型的“TTN”原间隔相邻基序（PAM）限制较少，最近受到了广泛关注。然而，由于其编辑效率相对较低，Cas12i3介导的多重基因编辑在植物中尚未见报道。在此，我们分别将包括T5E、UL12、PapE、ME15在内的四种5'核酸外切酶（Exo）与优化后的Cas12i3变体（Cas12i3-5M）的N端融合，并在水稻稳定株系中对六个内源性靶点系统评估了这些Exo:Cas12i3-5M融合体的编辑活性。我们证明，与Cas12i3和Cas12i3-5M相比，Exo:Cas12i3-5M融合体分别将基因编辑效率提高了12.46倍和1.25倍。值得注意的是，UL12:Cas12i3-5M融合体能够实现强大的单基因编辑，在六个测试的内源性基因中编辑效率高达90.42%-98.61%。我们进一步证明，尽管所有Exo:Cas12i5-5M融合体都能够进行多重基因编辑，但UL12:Cas12i3-5M在水稻稳定株系中同时编辑三个、四个、五个或六个基因时表现出卓越性能，效率分别为82.76%、61.36%、52.94%和51.06%。我们共同系统评估了不同的Exo:Cas12i3-5M融合体，并确定UL12:Cas12i3-5M是水稻中用于单基因和多重基因编辑的更强有力的系统。开发一种替代的强大单基因和多重基因编辑系统将丰富植物基因组编辑工具集，并促进聚合农艺重要性状以改良作物。
