Engineering a robust Cas12i3 variant‐mediated wheat genome editing system

• DOI: 10.1111/pbi.14544
• Publication Date: 2024-12-18T05:40:57Z
• Authors: Wenxue Wang; Lei Yan; Jingying Li; Chen Zhang; Yubing He; Shaoya Li; Lanqin Xia
• Keywords: Trans-activating crRNA

Wheat (Triticum aestivum L., 2n = 6x 42, AABBDD) is one of the most important food crops in world. CRISPR/Cas12i3, which belongs to type V-I Cas system, has attracted extensive attention recently due its smaller protein size and less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, relatively lower editing efficacy plants hexaploidy complex nature wheat, Cas12i3/Cas12i3-5M-mediated genome wheat not been documented yet. Here, we report engineering a robust Cas12i3-5M-mediated system through fusion T5 exonuclease (T5E) combination with an optimised crRNA expression strategy (Opt). We first showed that T5E, rather than ExoI, Cas12i3-5M increased gene efficiencies by up 1.34-fold 3.87-fold, compared Cas12i3 HEK293T cells, respectively. efficiency remains low wheat. then demonstrated Opt-T5E-Cas12i3-5M could enhance 1.20- 1.33-fold 4.05- 7.95-fold stable lines Opt-Cas12i3-5M Opt-Cas12i3, respectively, progressive 5'-end resection DNA strand at cleavage site deletion size. The enabled ranging from 60.71% 90.00% across four endogenous target genes three elite Chinese varieties. Together, developed enriches toolkits for either biological research or genetic improvement may be extended other polyploidy crop species.