Base editors for citrus gene editing

Abstract

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis ‘Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.

Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

“…CRISPR/Cas mediated genome editing is deemed the most promising approach to breed new citrus cultivars, owing to its short time requirement, precise genetic improvement and predictable results (Chen et al, 2019;Gao, 2021;Cao et al, 2022;Huang et al, 2022a). To date, SpCas9/gRNA from Streptococcus pyogenes, SaCas9/gRNA from Staphylococcus aureus, LbCas12a/crRNA from Lachnospiraceae bacterium and base editor derived from SpCas9/gRNA have been successfully adapted to modify citrus genomes for gene function study and new citrus cultivar breeding (Jia and Wang, 2014a;Jia et al, 2016;Peng et al, 2017;Zhang et al, 2017;Jia et al, 2017a, Jia et al, 2017bLeBlanc et al, 2018;Zhu et al, 2019;Jia et al, 2019a;Dutt et al, 2020;Huang et al, 2020;Jia and Wang, 2020;Huang and Wang, 2021;Jia et al, 2021;Alquezar et al, 2022;Jia et al, 2022;Mahmoud et al, 2022;Parajuli et al, 2022;Yang et al, 2022;Huang et al, 2022bHuang et al, , 2023Su et al, 2023).…”

“…For instance, canker-resistant Duncan grapefruit was developed through editing CsLOB1 coding region using SpCas9/gRNA (Jia et al, 2017b). Moreover, canker-resistant Duncan grapefruit, Hamlin, Pummelo and Wanjincheng orange were created by disrupting EBE pthA4 or the TATA box of CsLOB1 using spCas9/gRNA, LbCas12a/crRNA and base editor (Peng et al, 2017;Jia and Wang, 2020;Jia et al, 2022;Huang et al, 2022b). However, it must be kept in mind that all of the aforementioned canker-resistant genome-edited citrus plants are transgenic, which have not been commercialized owing to regulations and public perception concerns (Jia et al, 2006;Gong et al, 2021;Turnbull et al, 2021).…”

“…Multiple strategies have been developed to produce transgene-free plants after target-gene editing with CRISPR/Cas (Kocsisova and Coneva, 2023). Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins (Woo et al, 2015;Malnoy et al, 2016;Subburaj et al, 2016;Svitashev et al, 2016;Liang et al, 2017;Andersson et al, 2018;Su et al, 2023), novel delivery vectors such as viruses (Mei et al, 2019;Ma et al, 2020;Liu et al, 2023), unconventional selection methods to bypass integration of transgenes (Veillet et al, 2019;Alquezar et al, 2022;Huang et al, 2022bHuang et al, , 2023Wei et al, 2023), graft-mobile editing systems (Yang et al, 2023), and so on. Initially, Alquezar et al and Huang et al independently developed transgene-free citrus using CBE-mediated editing (Alquezar et al, 2022;Huang et al, 2022b).…”