Publications

Validation of endogenous U6 promoters for expanding the CRISPR toolbox in Nicotiana tabacum

Pan Luo, Siyuan Li, Lulu Li, Yanyan Li, Ying Qiao, Xianzhi Wu, Meiling Wu, Zhihao Song, Yong Yang, Ji Feng, Xingchun Tang

Abstract

In the past decade, the clustered regularly interspaced short palindromic repeat paired associated protein 9 (CRISPR-Cas9) mediated gene editing has been extensively utilized for investigation of gene function and the improvement of agronomic traits in plants (Zhu et al. 2020). The engineered CRISPR-Cas9 comprises two integral components: the Cas9 nuclease and a singular guide RNA (sgRNA). The sgRNA is a fusion of two RNA molecules, namely the spacer-containing CRISPR RNA (crRNA) and the transactivating crRNA (tracrRNA) that is essential for the maturation of the former moiety (Zess and Begemann 2021). For the recognition and cleavage of a target DNA site to occur, the sequence must also encompass a specific short motif, referred to as the protospacer adjacent motif (PAM), situated either distally or proximally (Zess and Begemann 2021). In addition, sgRNAs have been extensively designed and engineered with desirable functions for genome editing performance (Dong et al. 2022). U6 small nuclear RNAs (snRNA), which are non-coding RNAs, partake in the splicing of introns during the production of mature mRNAs in eukaryotic cells (Miyagishi and Taira 2002). U6 is synthesized by RNA polymerase III (Pol III), unlike the other spliceosomal snRNAs that are produced by RNA polymerase II (Didychuk et al. 2018). Consequently, the U6 promoter is frequently employed for the high-level expression of small RNAs and is the preferred choice for driving sgRNA expression in the CRISPR-Cas9 system (Friedland et al. 2013). Species-specific CRISPR-Cas9 systems have been formulated and applied in diverse plant species with gene-editing efficiencies exhibiting considerable variation (Zhu et al. 2020). Utilizing endogenous U6 promoters of various species to drive sgRNA has proven to be an effective and fruitful strategy for the CRISPR system (Long et al. 2018; Massel et al. 2022). Several studies have confirmed that using species-specific U6 promoters can increase sgRNA expression levels, thereby improving editing efficiency (Sun et al. 2015; Ng and Dean 2017). Up to the present, successful establishment of the CRISPR-Cas9 system using native U6 promoters has been achieved in Arabidopsis, cotton, rice, banana, and other species (Mohan et al. 2022; Zhang et al. 2022).

In the model tetraploid crop tobacco (Nicotiana tabacum), previous research has demonstrated the high efficiency of single-target editing driven by the AtU6-26 promoter in Arabidopsis (Gao et al. 2015). The mutation percentages are 81.8% for NtPDS and 87.5% for NtPDR6 (Gao et al. 2015). However, compared to the individual gRNA, the duplex gRNA driven by AtU6-26 promoter demonstrates lower efficiency (6.0 to 7.7%) in inducing indel mutations (Gao et al. 2015). In addition, N. tabacum is an allotetraploid derived from ancestors of the modern diploids N. sylvestris and N. tomentosiformis (Leitch et al. 2008). Thus, it is crucial to broaden the CRISPR toolbox for multi-target gene editing to effectively knock out target genes from the two sub-genomes. However, the current range of CRISPR tools accessible for N. tabacum remains limited due to its reliance on Arabidopsis U6 promoter (Mercx et al. 2016; Shi et al. 2019; Donovan et al. 2020; Tian et al. 2020, 2021; Li et al. 2021). For expanding the CRISPR toolbox in N. tabacum, three endogenous NtU6 promoters were reported and the effect of NtU6 promoter-driven CRISPR-Cas9 system within the tobacco plant was validated.