Highly efficient transgene-free genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR. (January 2023)

• Record Type: Journal Article
• Authors: Zhang, Jianduo; Xing, Jiaxin; Mi, Qili; Yang, Wenwu; Xiang, Haiying; Xu, Li; Zeng, Wanli; Wang, Jin; Deng, Lele; Jiang, Jiarui; Yang, Guangyu; Gao, Qian; Li, Xuemei
• Is Part Of: Plant science. Volume 326(2023)
• Publication Date: 2023-01
• Subjects: CRISPR/Cas9 -- OsU3-tRNA -- Transgene-free -- Ros1 -- NtLHT1; Botany -- Periodicals; Botanique -- Périodiques; 580
• DOI: 10.1016/j.plantsci.2022.111523
• Languages: English
• ISSNs: 0168-9452

Abstract

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. Highlights: In the manuscript, we provide an improved effective CRISPR/Cas9 system, pOREU3TR, for tobacco genome editing by the use of an OsU3-tRNA promoter combination to drive sgRNA expression, leading to greater mutagenesis efficiency compared with that of the existing system, pORE-Cas9 and a visible Ros1 expression cassette, AtUb10-Ros1 to monitor transgenic events, simplifying and increasing the efficiency of the screening procedure. The efficacy of this system was verified by editing an amino acid transporter ( NtLHT1 ) gene in tobacco. A lack of NtLHT1 function affected the plant height and both the transport and the distribution of amino acids in the leaves, demonstrating the high efficiency of the new editing system. Thus, This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements to increase the efficiency of plant genome editing.