Jianduo Zhang, Jiaxin Xing, Qili Mi. Wenwu Yang, Haiying Xiang, Li Xu, Wanli Zeng, Jin Wang, Lele Deng, Jiarui Jiang, Guangyu Yang, Qian Gao, Xuemei Li

Plant Science; Volume 326, January 2023, 111523

Available online 9 November 2022

Abstract

CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T₁ generation as a result of editing the NtLHT1 gene in tobacco, and the plant height and the contents of most free amino acids in the leaves of the T₂ mutant plants were significantly different from those in the leaves of WT plants, demonstrating the high efficiency of the new editing system. This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements for increasing the efficiency of plant genome editing.

See https://www.sciencedirect.com/science/article/pii/S016894522200348X

Fig. 1. Targeted mutations generated using OsU3-tRNA, AtU6-tRNA and AtU6 promoters. A Schematic illustration of pORE-Cas9 vectors. Synthesized OsU3-tRNA and AtU6-tRNA promoters were used instead of the AtU6 promoter to drive the expression of PDS sgRNA, and the 2×35S promoter was used to drive the expression of Streptococcus pyogenes Cas9 (SpCas9). NPTII, neomycin phosphotransferase gene; LB, left border; RB, right border. B Resistant calli containing OsU3-tRNA-PDS, AtU6-tRNA-PDS and AtU6 vectors generated on selective media. C Relative expression of tobacco PDS sgRNA driven by the three promoters in T0 plant leaves. D PCR identification of T-DNA insertions in the resistant calli using Cas9-specific primers. The CRISPR vector and WT calli were used as positive (P) and negative controls, respectively. M, DNA marker. a1-a9, b1-b9 and c1-c9 represent samples from OsU3-tRNA-PDS-, AtU6-tRNA-PDS-, and AtU6-PDS-containing calli, respectively. E Overview of the mutation rates of the calli for the PDS gene using the three vectors in tobacco. HD, wild-type Hong Hua Da Jin Yuan. J. Zhang et al.