Sweet orange is one of the most popular fruit crops worldwide. Traditional breeding approaches in sweet orange is impracticable due to the apomixis and long juvenility, making it difficult to obtain hybrids and selection of ideal genotypes. The development of targeted genome engineering technologies made it possible for the precise modification of target genes. Recently, a more efficient gene editing tool has been emerged based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system (Bhaya et al. 2011). The development of CRISPR/Cas9 technology is promising to accelerate the process of genetic improvement in perennial crops.
Gene editing in sweet orange are usually faced with difficulties including low editing efficiency and low root formation rate, which severely limits the advancement of gene function studies and application in molecular breeding in sweet orange. Recently, the application of Arabidopsis YAO promoter driven CRISPR/Cas9 system in citrus rootstock cultivar Carrizo Citrange significantly improved gene editing efficiency than the 35S promoter driven CRISPR/Cas9 system (Jia and Wang 2014; Zhang et al. 2017; Alvarez et al. 2021). Meanwhile, studies have shown that polycistronic tRNA-sgRNA (PTG) /Cas9 is more efficient for multiplex gene editing than traditional CRISPR/Cas9 system (Xie et al. 2015; Wang et al. 2018), and for Carrizo Citrange (Huang et al. 2020). Thus, it is possible to further improve the editing efficiency by adding the PTG to the YAO-driven Cas9 system in sweet orange. In addition, improving the surviving rate of transgenic sweet orange seedlings by avoiding the high risk of root induction process still requires further investigation (Belide et al. 2011). All those limitations impeding the process of generating healthy sweet orange plants with targeted genome editing using CRISPR/Cas9 system. To overcome those limitations, we developed a reliable protocol for the fast and efficient gene-edited sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90% (Fig. 1l). In addition, the gene editing efficiency was significantly improved by short-term heat stress treatment (Fig. 1o). Thus, our strategies provided a reference for the fast and efficient multiplex genome editing of sweet orange.
To further explore the application of PTG/Cas9 system in sweet orange, three gRNAs targeting phytoene desaturase (PDS) in Anliu sweet orange were used (Fig. 1a) (Jia and Wang 2014; Zhang et al. 2017). The three targets were assembled into pCAMBIA1300-pYAO:Cas9-eGFP vector (Fig. 1b). Statistical analysis revealed that the editing efficiency was about 36.7%-50% by Hi-Tom sequencing (Fig. 1c), and the albino phenotype can be precisely induced using the PTG/Cas9 system after 1 to 2 months (Fig. 1d). Sanger sequencing revealed that most of the mutations induced by PTG/Cas9 in sweet orange were small InDels(Fig. 1e-f, Fig. S1).
Genetic transformation in sweet orange has been vastly studied, and the transformation efficiency varies among sweet orange varieties. In this study, we optimized the transformation conditions by using vacuum negative pressure with the Agrobacterium-mediated genetic transformation of epicotyls for 5 min by using Anliu genotype (Fig. S2a). The transformation efficiency was 18.22%-21.15% (Fig. S2b). In order to shorten the time and improve the survival rate of transgenic sweet orange, transgenic seedlings were grafted on the sweet orange rootstocks in vitro (Fig. 1g-k). Our data showed that the survival rate of cleft grafting was more than 90% (Fig. 1l). As previous studies illustrated that heat stress could increase targeted mutagenesis induced by CRISPR/Cas9. In order to improve the mutation efficiency of grafted mosaic sweet orange (Fig. 1m), 10-month old grafted mosaic plants were exposed to short-term heat stress treatments at 37°C. We observed new albino tissues grown from mosaic seedlings 15 d after heat stress treatments (Fig. 1n, S3c-d), as well as the increased targeted mutagenesis (Fig. 1o).
The endogenous tRNA processing system exists in almost all species, which can not only process and cleave multiple gRNAs in one sgRNA expression cassette, but also act as transcription enhancers to enhance the expression of gRNAs (Xie et al. 2015). In this study, we evaluated the editing efficiency induced by the three PTG/Cas9 constructs using Agrobacterium-mediated genetic transformation of sweet orange epicotyls. For the target sites, the majority of the mutated alleles identified consisted of some deletions and therefore caused a shift of reading frame, while the mutation mediated by PTG/Cas9 system was a little different with YAO promoter-driven CRISPR/Cas9 system (Zhang et al. 2017). Meanwhile, we examined the off-target effects of the three PTG/Cas9 constructs, and no off-target mutations were detected in this study (primers used for amplification was listed in Table S1). Here, the editing efficiency of sweet orange induced by the three PTG/Cas9 vectors was not as efficient as the Carrizo Citrange (Zhang et al. 2017). We inferred that this may be related with the expression specificity of the YAO promoter (Li et al. 2010) and a single AtU6 promoter was used to drive the expression of several gRNAs, which requires further processing. The gene-edited seedlings in this study were directly induced from stem sections in 1-2 months (Fig. S2a), while the transgenic plants of Carrizo Citrange were regenerated from callus, in which the prolonged YAO promoter activity may modulate the expression of SpCas9 (Zhang et al. 2017). In addition to promoters, temperature could also regulate the activity of SpCas9 and the mutation efficiency (LeBlanc et al. 2018). We found that the the mutation efficiency of chimera sweet orange plants was also significantly increased after heat stress treatments (Fig. 1o), our result was consistent with previous report in Arabidopsis and Carrizo Citrange (LeBlanc et al. 2018).
Cleft grafting in vitro could significant improve the survival rate of transgenic seedlings, and this was a suitable method for those plants which are hardly to root or susceptible to various soil pathogenic bacteria (Belide et al. 2011). The combination of grafting and heat stress treatment may be a suitable method for the knockout of plant reproduction- and development-related genes, which may affect plant regeneration or rooting if they are mutated at early growth stages. Here, we developed a fast and efficient gene editing method for sweet orange, our approach offers a new way to facilitate the efficient multiplex gene editing of sweet orange, and this approach is especially suitable for the genetic improvement of citrus varieties.